微生物学

Cyclic diadenosine monophosphate (c-di-AMP) is a recently discovered second messenger that modulates several signal transduction pathways in bacterial and host cells. Besides the bacterial system, c-di-AMP signaling is also connected with the host cytoplasmic surveillance pathways (CSP) that induce type-I IFN responses through STING-mediated pathways. Additionally, c-di-AMP demonstrates potent adjuvant properties, particularly when administered alongside the Bacillus Calmette–Guérin (BCG) vaccine through mucosal routes. Because of its pivotal role in bacterial signaling and host immune response, this molecule has garnered significant interest from the pharmaceutical industry. This protocol outlines the quantification of c-di-AMP by an HPLC-based assay to enumerate the activity of c-di-AMP synthase from Mycobacterium smegmatis. The following protocol is designed to be generic, enabling the study of c-di-AMP synthase activity from other bacterial species. However, modifications may be required depending on the specific activity of c-di-AMP synthase from different bacterial sources.

Cryo-electron microscopy (cryo-EM) is a powerful technique capable of investigating samples in a hydrated state, compared to conventional high-vacuum electron microscopy that requires samples to be completely dry. During the drying process, numerous features and details may be lost due to damage caused by dehydration. Cryo-EM circumvents these problems by cryo-fixing the samples, thereby retaining the intact and original features of hydrated samples. This protocol describes a step-by-step cryo-scanning electron microscopy (cryo-SEM) experimental procedure with Chlorella sorokiniana as the subject. By employing filter paper as the sample substrate, we propose a simple and reliable method for cryo-fixation and freeze-fracture of Chlorella sorokiniana in water suspension. The advantage of using filter paper as a substrate lies in its ability to support a thin film of sample, enabling a cold knife to make a cut effortlessly and produce a clean freeze-fractured surface for SEM investigation. By following the approach described in this protocol, both the internal structure and surface morphology of Chlorella sorokiniana can be easily resolved with high quality. This protocol is highly versatile and can be applied to samples dispersed in water or solvents, including cyanobacterial cells, algal cells, and any kind of sample that can be adsorbed onto filter paper.

Droplet microfluidic platforms have been broadly used to facilitate DNA transfer in mammalian and bacterial hosts via methods such as transformation, transfection, and conjugation, as introduced in our previous work. Herein, we recapitulate our method for conjugal DNA transfer between Bacillus subtilis strains in a droplet for increased conjugation efficiency and throughput of an otherwise laborious protocol. By co-incubating the donor and recipient strains in droplets, our method confines cells into close proximity allowing for increased cell-to-cell interactions. This methodology is advantageous in its potential to automate and accelerate the genetic modification of undomesticated organisms that may be difficult to cultivate. This device is also designed for modularity and can be integrated into a variety of experimental workflows in which fine-tuning of donor-to-recipient cell ratios, growth rates, and media substrate concentrations may be necessary.

Carbohydrates serve crucial functions in most living cells, encompassing structural and metabolic roles. Within the realms of plant and algal biology, carbohydrate biosynthesis and partitioning play pivotal roles in growth, development, stress physiology, and various practical applications. These applications span diverse fields, including the food and feed industry, bioenergetics (biofuels), and environmental management. However, existing methods for carbohydrate determination tend to be costly and time-intensive. In response to that, we propose a novel approach to assess carbohydrate partitioning from small samples. This method leverages the differential solubility of various fractions, including soluble sugars, starch, and structural polymers (such as cellulose). After fractionation, a straightforward spectrophotometric analysis allows for the quantification of sugars.

The quality of cellular products used in biological research can impact the accuracy of results. Epstein–Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. Existing tests to detect EBV can be divided into three categories: nucleic acid assays, serological assays, and in situ hybridization assays. However, most methods are time-consuming, expensive, and not conducive to high-volume clinical screening. Therefore, a simple system that allows for the rapid detection of EBV in multiple contexts, including both cell culture and tissue samples, remains necessary. In our research, we developed EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 10³ copy numbers for the RPA-based and RPA-LFA systems and 1 × 10⁴ copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those of the assays specified in industry standards. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

In modern science, the exchange of scientific material between different institutions and collaborating working groups constitutes an indispensable endeavor. For this purpose, bacterial strains are frequently shipped to collaborators to advance joint research projects. Bacterial strains are usually safely shipped as cultures on solid medium, whereas the shipment of liquid cultures requires specific safety measures due to the risk of leakage. Cyanobacterial cultures are frequently maintained as liquid stock cultures, and this problem typically arises. This protocol describes a new method for the shipment of liquid cyanobacterial stock cultures by agarose gel embedding (SCAGE). More specifically, a cyanobacterial culture is mixed with low-melting agarose and cast into sterile plastic bags, resulting in a thin, solid cyanobacterial agarose gel (cyanogel) that can be easily shipped. After delivery, subsequent regeneration of the cyanogel material in liquid media results in full recovery of the examined bacterial strains. Thus, the packaging method devised in the present study comprises an innovative technique to facilitate the shipment of bacterial strains, whilst eliminating previously encountered issues like cell culture leakage.

This protocol outlines the use of the previously described sodium hypochlorite extraction method for estimating the accumulation of polyhydroxybutyrate (PHB) in bacteria. Sodium hypochlorite (NaClO) is widely used for PHB extraction as it oxidizes most components of the cells except PHB. We assessed the feasibility of using NaClO extraction for the estimation of PHB accumulation in bacterial cells (expressed as a percentage w/w). This allowed us to use a simple spectrophotometric measurement of the turbidity of the PHB extracted by NaClO as a semiquantitative estimation of PHB accumulation in the marine microorganisms Halomonas titanicae KHS3, Alteromonas sp., and Cobetia sp. However, this fast and easy protocol could be used for any bacterial species as long as some details are considered. This estimation exhibited a good correlation with the accumulation measured as dry cell weight or even with the accumulation measured by crotonic acid and HPLC quantifications. The key advantage of this protocol is how fast it allows an estimation of PHB accumulation in Halomonas, Alteromonas, and Cobetia cultures (results are available in 50 min), enabling the identification of the appropriate moment to harvest cells for further extraction, polymer characterization, and accurate quantification using more reliable and time-consuming methods. This protocol is very useful during bacterial cultivation for a quick evaluation of PHA accumulation without requiring (i) large volumes of cultures, (ii) a long time for analysis compared to dry cell weight, (iii) preparation of standard curves with sulfuric acid hydrolysis for crotonic acid quantification, or (iv) specific equipment and/or technical services for HPLC quantification.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1–4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1–4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS).

Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from Acinetobacter genomospecies 16 (A. gp16), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments.

Candida albicans is the most common human fungal pathogen, able to reside in a broad range of niches within the human body. Even though C. albicans systemic infection is associated with high mortality, the fungus has historically received relatively little attention, resulting in a lack of optimized molecular and fluorescent tools. Over the last decade, some extra focus has been put on the optimization of fluorescent proteins (FPs) of C. albicans. However, as the FPs are GFP-type, they require an aerobic environment and a relatively long period to fully mature. Recently, we have shown the application of a novel type of fluorogen-based FP, with an improved version of fluorescence activating and absorption shifting tag (iFAST), in C. albicans. Due to the dynamic relation between iFAST and its fluorogens, the system has the advantage of being reversible in terms of fluorescence. Furthermore, the combination of iFAST with different fluorogens results in different spectral and cellular properties, allowing customization of the system.