生物科学

Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany). Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments.

Key features

• Quantification of wood decay ability of mixed cultures.

• Allows testing if fungi are more efficient in degrading wood when bacteria are present.

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.

Bacterial swarming refers to a rapid spread, with coordinated motion, of flagellated bacteria on a semi-solid surface (Harshey, 2003). There has been extensive study on this particular mode of motility because of its interesting biological and physical relevance, e.g., enhanced antibiotic resistance (Kearns, 2010) and turbulent collective motion (Steager et al., 2008). Commercial equipment for the live recording of swarm expansion can easily cost tens of thousands of dollars (Morales-Soto et al., 2015); yet, often the conditions are not accurately controlled, resulting in poor robustness and a lack of reproducibility. Here, we describe a reliable design and operations protocol to perform reproducible bacterial swarming assays using time-lapse photography. This protocol consists of three main steps: 1) building a “homemade,” environment-controlled photographing incubator; 2) performing a bacterial swarming assay; and 3) calculating the swarming rate from serial photos taken over time. An efficient way of calculating the bacterial swarming rate is crucial in performing swarming phenotype-related studies, e.g., screening swarming-deficient isogenic mutant strains. The incubator is economical, easy to operate, and has a wide range of applications. In fact, this system can be applied to many slowly evolving processes, such as biofilm formation and fungal growth, which need to be monitored by camera under a controlled temperature and ambient humidity.

Characterization of biofilm formation and metabolic activities is critical to investigating biofilm interactions with environmental factors and illustrating biofilm regulatory mechanisms. An appropriate in vitro model that mimics biofilm in vivo habitats therefore demands accurate quantitation and investigation of biofilm-associated activities. Current methodologies commonly involve static biofilm setups (such as biofilm assays in microplates, bead biofilms, or biofilms on glass-slides) and fluidic flow biofilm systems (such as drip-flow biofilm reactors, 3-channel biofilm reactors, or tubing biofilm reactors). Continuous flow systems take into consideration the contribution of hydrodynamic shear forces, nutrient supply, and physical transport of dispersed cells, which define the habitat for biofilm development in most natural and engineered systems. This protocol describes the assembly of 3 flow-system setups to cultivate Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1 model biofilms, including the respective quantitation and observation approaches. The standardized flow systems promise productive and reproducible biofilm experimental results, which can be further modified according to specific research projects.