The current investigation utilises an array of assessment techniques including the following: culture media, reagents, lectin-carbohydrate interactions, and bacterial strains of interest for the analysis of cell surface features. As a result, coaggregation experiments can be performed via spectrophotometric measurement, confocal microscopy, Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and a quantitative spectrophotometric assay. In order to identify coaggregation, experiments typically include combining several bacterial strains of interest and examining any physical interactions, such as clumps, under a microscope or by detecting changes in light scattering using a spectrophotometer. The optical density of the bacterial suspension before and after coaggregation was determined, resulting to data in terms of coaggregative index or percent flocculation. In this way, the extent of coaggregation can be measured. The function of lectin-carbohydrate interactions in coaggregation may be studied using heating and sugar-addition studies. Bacteria that have been heated to death can be employed as negative controls, and sugar solutions can be utilized to prevent coaggregation by preventing interactions between lectin and carbohydrates. Coaggregation investigations often combine microscopy, quantitative analysis, and bacterial culture techniques to investigate into the physical and molecular processes behind bacterial cell-cell interactions.
1. Bacterial strains and culture conditions
The oral bacteria strains S. gordonii ATCC 35105, A. oris, F. nucleatum, S. oralis and V. parvula additionally, four enteric bacterial strains were used: Clostridium difficile, Enterococcus faecalis, Enterococcus faecium, Escherichia coli were utilized in this investigation. 1.5% w/v bacto-agar (Melford Laboratories Ltd., Suffolk, UK) has been supplied following sterilizing for all solid-medium-grown bacteria. Moreover, liquid and solid media were sterilized by autoclaving at 121°C and 15 pressure for 15 min. Regular cultures of S. gordonii and A. oris were carried out in 20 mL Todd Hewitt Yeast Extract (THYE) medium, which included 5 g/L Yeast Extract and 30 g/L Todd Hewitt Broth (Difco, Detroit, MI) (Sigma-Aldrich, UK). In order to cultivate F. nucleatum 25586, FAA has been used, which included 46 g/L Fastidious Anaerobic Broth (FAB; Oxoid, Leicester, UK), 2.5 g/L L-glutamic Acid, (w/v) Neopeptone, and 5 g/L yeast extract (all Sigma-Aldrich, UK). V. parvula, Clostridium difficile, Enterococcus faecalis, Enterococcus faecium, Escherichia coli were typically cultivated in BHYGL medium, which contained 14 mL of 88% (v/v) DL-lactate, 5 g/L of yeast extract, 2.5 g/L of L-glutamic acid monosodium salt hydrate, and 37 g/L of brain Heart Infusion (Melford Laboratories Ltd., UK) (all Sigma-Aldrich, UK). At 37°C and 90% N2-5% H2-5% CO2 saturation, cells were grown anaerobically in a Bugbox Plus anaerobic workstation (Baker Ruskinn, Bridgend, UK) for 18–48 hours without shaking. Before autoclaving, the pH of the V. parvula culture was adjusted with NaOH to pH 7.5. All media were created using de-ionized water.
2. Preparation of inoculate for coaggregation assays:
Agar plates were used to cultivate bacterial strains overnight before they were transferred to a liquid media to continue growing. Depending on the kind of bacteria, the liquid cultures were either incubated under anaerobic or aerobic conditions for 18–24 hours at 37°C and then bacterial cells were separated by centrifugation and then washed twice via phosphate buffered saline (PBS- pH of 7.2). This step was significant and presumably made to ensure and guarantee that the bacterial cells were uniform and pure and it also aided in removing any such extra component which would have complicated future investigations. The final cell density for all the strains in PBS was adjusted to near 108 cells per ml and then these bacterial suspension-containing inocula were used in coaggregation assays.
3. Coaggregation assay
The quantitative spectrophotometric test procedure used in this research was a slight modification of the procedure outlined by Ikegami et al. in 2004. This test is based on the premise that cell clusters developed during coaggregation leads to a decrease in optical density of bacterial suspension. Prior to the procedure, firstly bacteria were centrifuged at 10,00g for five minutes and then resuspended in coaggregation solution composed of calcium magnesium sodium chloride Tris-buffer. (This investigation used the coaggregation solution, which has 1 mM Tris buffer with a pH 7.0 adjustment, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.15 M NaCl, and 3.1 mM NaN3). The bacterial cultures were again diluted to a final OD60 nm of 1.5 and combined in sterilized cuvettes equally. To determine coaggregation, the optical densities of mixes were determined before and after an hour incubation at room temperature. To determine the % coaggregation, use the following formula: (OD before mixing - OD after mixing) / (OD before mixing - OD before mixing) x 100, this formula determines the degree of coaggregation between bacterial strains.
Percent coaggregation = (Optical density before mixing – Optical density after mixing) / Optical density before mixing x 100
The formula above was usedto determine the percentage of coaggregation between two bacterial strains. The optical density (OD) of the bacterial strains before to mixing is referred to as the pre-incubation value. The test value is the OD of the bacterial strains following their mixing and one-hour coaggregation. The test value OD is subtracted from the pre-incubation value OD in the numerator before being divided by it. To represent the coaggregation as a percentage, the result of dividing (OD before mixing - OD after mixing) by OD before mixing is multiplied by 100. This makes it possible to compare coaggregation across various bacterial strains and in various environments.
The same technique had been employed to quantify autoaggregation, or the ability of a single bacterial strain to aggregate, by mixing two equal volumes of the same bacterial solution. In this experiment, a concentration of 500 mL of each solution with an OD600 nm of 1.0 was used to combine a number of strains. Coaggregation was assessed after a 2-minute incubation time at room temperature using a scoring method created by Kolenbrander in 1995 (17). According to this scoring system, scores range from 0 to 100, with 100 representing the quick and rapid dropping of massive coaggregates.
4. Influence of heating on coaggregation
To investigate the influence of heating on coaggregation, the following steps have been used: bacterial suspensions were heated at 70°C for 10 min, then cooled on ice for 5 min before coaggregation was tested. The suspensions that had undergone heat treatment were contrasted with suspensions that had undergone no heating at all (one cuvette was maintained at room temperature while the other was submerged in a water bath heated to 60°C for 30 minutes). The cuvette was heated, brought back to room temperature, and the OD600 nm was measured to determine the degree of coaggregation. The same methodology as previously described was used to evaluate coaggregation, using a score system based on coaggregate settling and supernatant clarity. After three iterations of the experiment, the data were averaged and compared to determine the effect of heating on coaggregation strengt
5. Coaggregation imaging and Confocal scanning laser microscopy analysis
Picogreen was used to stain bacterium cells at a concentration of 5 M (15 ng/ml), whereas DAPI, propidium iodide (red) stain were used to stain A. oris, F. nucleatum, S. oralis and V. parvula cells at a concentration of 30 M (20 g/ml). After labeling, cells were incubated individually for 1–2 hours at 37°C. Bacteria have been three times washed with coaggregation buffer before being mixed for coaggregation. Equivalent quantities (300 l) of the partner cells and S. gordonii were introduced to a glass test tube, vortexed for 10 seconds, and then gently rocked until coaggregation was visible. 1.0 mm diameter rubber O rings were attached to glass microscope slides using sticky wax or superglue in the initial attempts to image coaggregates using Confocal scanning laser microscopy (CSLM). A cell chamber setup and a coverslip were subsequently employed. In the center of the rubber ring, coaggregated cells or monoculture controls were placed, mixed well, and then a coverslip was attached. A Leica SP2 CLSM microscope (Leica, Microsystems, Heidelberg, Germany) was used to view coaggregation samples. Excitation (Ex) at 530 nm and emission (Em) at 630 nm for propidium iodide and Ex/Em = 485 nm/530 nm for Picogreen were used.
6. Transmission electron microscopy (TEM)
Coaggregation samples were centrifuged for 1 min at 1000 g, deposited immediately after coaggregation into 2% glutaraldehyde in 100 mM phosphate buffer, pH 7.0, and kept at 4°C for high-resolution imaging of coaggregation by TEM. Samples were fixed in an epoxy resin and sectioned at Newcastle University's Electron Microscopy Research Services after being dehydrated through a graded series of ethanol washes (Kalab et al., 2008). Using a Philips CM100 transmission electron microscope, sections were examined.
7. Create a 3D reconstruction of coaggregation (SEM)
To obtain the three-dimensional ultrastructure of coaggregates generated between oral bacteria, scanning electron microscopy (SEM) was utilized. Cells were extracted after coaggregation (Section 2.4.1) at 1,000 g, 4°C for 1 min. After discarding the supernatant, the cells were collected and fixed in 2% (v/v) glutaraldehyde at 4°C for 16–24 hours. After being twice washed in PBS, the cells underwent a series of ethanol washes to dehydrate them: 25% ethanol for 30 minutes, 50% ethanol for 30 minutes, 75% ethanol for 30 minutes, and two washes for 1 hour in 100% ethanol. The coaggregation blocks have been cut to 0.75 mm x 0.75 mm, and afterwards, polyvinyl chloride was used to bind them to a pin (glue). With the use of the Gatan 3 View and Sigma SEM (Zeiss, Cambridge, UK), around 120 sections with a 70 nm separation were collected (Gatan inc., Abingdon, UK). DM3 format was used to store the raw data.
8. Image processing
The essential step was to adjust the picture contrast, and then all photos were converted from DM3 to TIFF format using Microscopy Image Browser (MIB v6, Institute of Biotechnology, University of Helsinki, Finland). Gradient (2D/3D) filters were employed to minimize noise and contrast during the image processing processes across all photos. The next stage was coloring the interacting bacteria; during this procedure, each slice was manually categorized depending on the form or contrast of the bacterium. This information were utilized to create a 3D model of coaggregation. The above-mentioned sequence of consecutive photos were integrated into three-dimensional stacks using MIB in order to achieve the proper z-axis. A 3D model of coaggregation was made using this information. To get the correct z-axis, the previously described series of sequential pictures were combined into three-dimensional stacks using MIB.