Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies. The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesiculosus inside the chamber affected colonization of the outer membranes by marine bacteria.
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Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies.
The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesiculosus inside the chamber affected colonization of the outer membranes by marine bacteria. The alga did indeed enrich the total population of colonizing bacteria by more than a factor of four. These findings lead us to investigate the effect of the presence of the coccolithophoric alga Emiliania huxleyi on attachment and biofilm formation of the marine bacterium Phaeobacter inhibens DSM These organisms co-exist in the marine environment and have a well-characterized interdependence on secondary metabolites.
The experiment was carried out using a wild type wt strain as well as a TDA-deficient strain of P. The ability of the bacterium to produce the antibacterial compound, tropodithietic acid TDA influenced its attachment since the P. Whilst the attachment of the bacterium to a surface was facilitated by presence of the alga, however, we cannot conclude if this was directly affected by the algae or whether biofilm formation was dependent on the production of TDA by P.
In the light of these results, other applications of immersed culture chambers are suggested. Microbiologists have for decades been focused on isolation and growth of pure bacterial cultures.
To support these aims, techniques are being developed for microbial co-culture studies and these are also being used as means of increasing culturability of microorganisms from natural samples Kaeberlein et al.
Examples of such techniques include diffusion chambers such as the iChip Nichols et al. The membranes allow for nutrient, quorum sensing molecules and other small naturally occurring compound to diffuse into the chamber. Several variants of such culture chambers exist including the microbial culture chip, where bacteria grow on a membrane subdivided into hundreds of small compartments.
The membrane is placed on a liquid or an agar surface and micro-colonies develop in each compartment Ingham et al. Microbial culture chambers are generally single chambers connected to the environment by porous membranes Kaeberlein et al. These are built to enrich a complex mixture of microorganisms. Current designs are not orientated toward the formation of biofilms and do not allow for evaluation of why a specific population of microorganisms are cultured.
This device was designed to allow for experiments to be conducted within the natural environment with the focus on biofilm formation. Communities of marine bacteria are known to live in association with and colonize the surface of macro algae. Some algae such as Fucus vesiculosus , produce a range of chemical compound to attract a specific mixed bacterial community Wahl, ; Lachnit et al. Others live in more species specific interdependent relationships. We have, over the past decade, studied the interactions between the marine bacterium, Phaeobacter inhibens and other bacteria, especially those that are fish pathogenic Prol et al.
In the marine environment, P. The bacterium is able to switch from mutualist to parasite in response to the growth and life cycle of E. The relationship is mutualistic when the algae produce dimethylsulfoniopropionate DMSP , which provides a source of sulfur and carbon for the bacteria, and the bacteria produce growth hormones and anti-bacterial compounds for the algae in form of phenylacetic acid and TDA, respectively.
The relationship becomes parasitic, when the algae reach stationary phase and secrete p -coumaric acid pCA as well as sinapic acid, which are potential senescence signals, to which the bacteria react by activating otherwise silent biosynthetic pathways that encode the algaecidal roseobacticides and roseochelins, respectively Seyedsayamdost et al. Phaeobacter inhibens is an excellent biofilm former Bernbom et al.
We therefore speculate that the presence of E. To enable studies of such co-culture interactions, we developed and applied the microbial culture chamber and propose that it can also be used in future studies for enhancement and development of new antibacterial compounds.
For co-cultivation, we used the microbial culture chamber, a stainless-steel device for in situ culture and enrichment of microorganisms Figure 1. The culture chamber has a central chamber connected to the outside through a porous membrane supplied by the user so material and pore size is optional allowing microorganisms to be cultured on the external surface or the membrane with access to diffusible molecules from the inner chamber experimental membrane.
A second membrane is sealed from access to the chamber by a solid plate and acts as a control control membrane Figure 2. A second solid plate acts as a spacer and can be removed to accommodate thicker stacks of membranes, if required Figure 1. A thicker stack of membranes on the outer side of the chamber can be deployed and has the function of fine tuning the rate of diffusion between environment and inner chamber.
Schematics of the microbial culture chamber made from stainless steel, total height 5 cm. A Disassembled view. B View of assembled chamber with component numbers as described for panel A. C Cut away view of assembled chamber. A—C Set up of culture chamber from autoclaved components plus sterile membranes with numbering as indicated in Figure 1. A Add control membrane 11 using sterile tweezers and then at least one spacer 6 ensuring tight fit then fill central chamber with culture medium and, in the experiments described here, with E.
B Add experimental membrane 10 , avoiding air bubbles. C Screw down cap This set up allows for co-culturing of two organisms while keeping them physically separated by the membrane. Alternatively, this device makes it possible to fill the inner chamber with, e. The idea is that microorganisms from a given environment or pure culture will be able to attach and grow and form a biofilm on the outer surface of the membranes while being exposed to the outside environment as well as to compounds diffusing out from the inner chamber.
The solid plates can be supplemented with an exterior porous membrane to serve as a control, where the membrane is only exposed to the outside environment without being in contact with the inner chamber. The chamber components were autoclaved prior to assembly in a laminar flow hood Figure 2A and then the set is up completed, as described in Figure 2 and in individual experiments, below.
K02CP sealed from the inner chamber with two metal plates Figure 1. The inner chamber was filled with 1 ml 7-days culture of E. The experiment was carried out in two independent experiments, each in triplicate, and incubations were for 24, 48, and 96 h. Tracking beads 0. After incubation, the chambers were dissembled. Three experimental membranes and three control membranes were randomly selected for SYBR Gold staining to evaluate bacterial biofilm formation by epifluorescence microscopy.
In the same manner, three experimental membranes and three control membranes were selected for DNA extraction for subsequent qPCR to quantify the number of bacteria attached below. The algal fragment was washed in 5 ml of sterile artificial sea water. The assembled chamber was suspended in a low nutrient environment, i. The artificial sea water was spiked with 1 ml of medium used to wash the algal fragment. After a week, microorganisms from the outer surfaces of the membranes were then quantified after staining with SYTO9 and hexidium iodide and imaging as described below.
At the end of each experiment viable counts on L-agar were made for the bacteria in the central chamber to determine the degree to which the external antibiotics were affecting the growth of E. Additional experiments were performed in the absence of microorganisms using phosphate buffered saline; by loading the inner chamber with 1 mm fluorescein Sigma, NL. After incubation, P. A cover slip was mounted on top. A similar procedure was used for SYTO9 and hexidium iodide staining microorganisms on porous aluminum oxide membranes as previously described Ingham et al.
The primers used were designed within the 16S gene of P. All samples were subjected to melting curve analysis. Four individual cultures of P. Normality tests were performed in Minitab Minitab 18, Minitab Inc. Individual samples were compared using paired t -test confidence level: After incubation for a week, the outer surface of the PAO membranes both experimental and control membranes were colonized with microorganisms that could be visualized by staining with SYTO9 and hexidium iodide Figure 3.
Enrichment of the biofilm was observed on the open, experimental membrane relative to the outer surface of the PAO membrane without a connection to the central chamber the closed membrane. The enrichment factor was an average 4. This suggests that nutrients originating from F. This result suggested it would be possible to look at a more specific microbial interaction using the culture chamber, as described below. Randomly selected areas 2. Incubation of E. These data suggest that the two chemically unrelated antibiotics could enter the growth chamber at effective doses.
Additionally, the dye fluorescein, loaded into the chamber, was found to approach equilibration within 8 h with detectable fluorescein found after 1 h. The number of attached P.
There were more attached single cells and formation of large plaques on the outside of the experimental membranes Figure 4 , where P. A mixture of single cells and biofilm plaques were observed on both experimental and control membranes Figure 5. A Experimental membranes. B Control membranes. The attachment of P. After 24 and 48 h of incubation, the TDA-producing P. In contrast, the numbers of attached P.
This indicates that production of TDA affects initial attachment and biofilm formation, and that it over time becomes inhibitory for biofilm formation. Number of attached P. After 24 h of incubation, P. This indicates that the presence of E. There were no significant trends in attachment of P. One source of error during set up of the culture chambers was leakage due to improper assembly or cracked PAO membranes. Two approaches were taken to address with this. The first was the use of a 0.
The second was the use of microbeads as a control for leakage. When chambers were loaded with 0. Whilst the user will need to calibrate these cut-off value for themselves, depending on the membrane porosity and adherence properties, we recommend this approach to remove invalid outliers from data sets.
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