Biological Sciences

Over the past decades, the main techniques used to visualize bacteria in tissue have improved but are still mainly based on indirect recognition of bacteria. Both microscopy and molecular recognition are being improved, but most procedures for bacteria detection in tissue involve extensive damage. Here, we describe a method to visualize bacteria in tissue slices from an in vivo model of breast cancer. This method allows examining trafficking and colonization of fluorescein-5-isothiocyanate (FITC)-stained bacteria in various tissues. The protocol provides direct visualization of fusobacterial colonization in breast cancer tissue. Rather than processing the tissue or confirming bacterial colonization by PCR or culture, the tissue is directly imaged using multiphoton microscopy. This direct visualization protocol causes no damage to the tissue; therefore, all structures can be identified. This method can be combined with others to co-visualize bacteria, types of cells, or protein expression in cells.

The study of haloarchaea provides an opportunity to expand understanding of the mechanisms used by extremophiles to thrive in and respond to harsh environments, including hypersaline and oxidative stress conditions. A common strategy used to investigate molecular mechanisms of stress response involves the deletion and/or site-directed mutagenesis of genes identified through omics studies followed by a comparison of the mutant and wild-type strains for phenotypic differences. The experimental methods used to monitor these differences must be controlled and reproducible. Current methods to examine recovery of halophilic archaea from extreme stress are complicated by extended incubation times, nutrients not typically encountered in the environment, and other related limitations. Here we describe a method for assessing the function of genes during hypochlorite stress in the halophilic archaeon Haloferax volcanii that overcomes these types of limitations. The method was found reproducible and informative in identifying genes needed for H. volcanii to recover from hypochlorite stress.

Bacteriocins are antimicrobial peptides with activity against antibiotic resistant bacterial pathogens. Here, we describe a set of methods aimed at purifying, identifying, and characterizing new bacteriocins. The purification consists of ammonium sulphate precipitation, cation-exchange chromatography, and reversed-phase chromatography. The yield of the bacteriocin is quantified by bacteriocin antimicrobial activity in a microtiter plate assay after each purification step. The mass of the purified bacteriocin is assessed by MALDI TOF MS analysis of the active fractions after reversed-phase chromatography. The mass is compared with the theoretical mass based on genetic information from the whole genome sequencing of the bacteriocin producer strain. Physicochemical characterization is performed by assessing antimicrobial activity following heat and protease treatments. Fluorescent techniques are used to examine the capacity of the bacteriocin to disrupt membrane integrity. Herein a set of protocols for purification and characterization of the bacteriocin nisin Z is used as a typical example in this paper.

Bacterial studies based on growth curves are common in microbiology and related fields. Compared to the standard photometer and cuvette based protocols, bacterial growth curve measurements with microplate readers provide better temporal resolution, higher efficiency, and are less laborious, while analysis and interpretation of the microplate-based measurements are less straightforward. Recently, we developed a new analysis method for evaluating bacterial growth with microplate readers based on time derivatives. Here, we describe a detailed protocol for this development and provide the homemade program for the new analysis method.

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution.

Graphic abstract:

Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.

Basic and translational research needs rapid methods to test antimicrobial formulations. Bioluminescent bacteria and advanced imaging systems capable of acquiring bioluminescence enable us to quickly and longitudinally evaluate the efficacy of antimicrobials. Conventional approaches, such as radial diffusion and viable count assays, are time-consuming and do not allow for longitudinal analysis. Bioluminescence imaging is sensitive and gives vital spatial and temporal information on the infection status in the body. Here, using bioluminescent Pseudomonas aeruginosa, we describe an in vitro and an in vivo approach to rapidly evaluate the antimicrobial efficacy of the host-defense peptide TCP-25.

Graphic abstract:

Evaluation of antimicrobials using bioluminescent bacteria.

Caenorhabditis elegans is a ubiquitous free-living nematode that feeds on bacteria. The organism was introduced into a laboratory setting in the 1970s and has since gained popularity as a model to study host-bacteria interactions. One advantage of using C. elegans is that its intestine can be colonized by the bacteria on which it feeds. Quantifying the bacterial load within C. elegans is an important and easily obtainable metric when investigating host-bacteria interactions. Although quantification of bacteria harbored in C. elegans via whole-worm lysis is not a novel assay, there is great variation between existing methods. To lyse C. elegans, many protocols rely on the use of a hand-held homogenizer, which could introduce systematic error and subsequent variation between researchers performing the same experiment. Here, we describe a method of lysing the intestines of C. elegans to quantify the bacterial load within the intestine. Our method has been optimized for removing exogenous bacteria while maintaining worm paralysis, to ensure no bactericidal agents are swallowed, which could kill bacteria within the intestine and affect results. We utilize and compare the efficiency of two different homogenization tools: a battery-powered hand-held homogenizer, and a benchtop electric homogenizer, where the latter minimizes variability. Thus, our protocol has been optimized to reduce systematic error and decrease the potential for variability among experimenters.

Graphic abstract:

Simplified overview of the procedure used to quantify the bacterial load within C. elegans.

The two different methods are herein described for worm lysis: “Option 1” is a hand-held homogenizer, and “Option 2” is a benchtop homogenizer.

Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.

Fungal metallo-tolerance has been described in different species and plays an important role in bioremediation of contaminated environments. Metallo-tolerance is mainly documented by microdilution assays and agar well diffusion methods using equipment that can be expensive. The tolerance index can be calculated to determine the efficiency of a fungus to degrade and resist heavy metals. The present protocol is based on analyzing the tolerance index and minimum inhibitory concentration of the metallo-tolerance potential of culturable fungi on solid media. This can be calculated by daily measurements of colony size on agar supplemented with different concentrations of heavy metals. This method is an easy approach to determine fungal heavy metal resistance using simple laboratory equipment without spectroscopy.

Colloidal chitin (CC) is a common substrate used in research work involving chitin-active enzymes (chitinases). Cell free supernatant (CFS) is prepared from fermented broth. Preparation of CC and CFS usually involve large amounts of liquid, which must be separated from the solids. This necessitates the use of a large volume centrifugation facility, which may not be accessible to everyone. Filtration is a viable alternative to centrifugation, and several filter elements are described in the literature. Each of those elements has its own set of disadvantages like non-availability, high cost, fragility, and non-reusability. Here we describe the use of lab coat clothing material (LCCM) for the preparation of CC and CFS. For filtration purposes, the LCCM was found to be functional, rugged, reusable, and cost-effective. Also described here is a new method for the estimation of laminarinase using a laminarin infused agarose gel plate. An easily available optical fabric brightener (OFB) was used as a stain for the agarose plate. The laminarin infused agarose plate assay is simple, inexpensive, and was found to be impervious to high amounts of ammonium sulfate (AS) in enzyme precipitates.