Table 1.
Bacterial genera isolated and identified from cultures of follicular fluid.
Table 2.
Bacterial growth in hormone supplemented thioglycollate broth.
Figure 1.
Light micrographs of Gram stains and biofilm slides.
A–D (a)) Light microscopy image at ×1000 total magnification of Gram stained bacterial S. agalactiae and Lactobacillus spp. colonies cultured from biofilms. (A–D (b)) Light microscopy image at × 1000 total magnification of Congo red stained S. agalactiae and Lactobacillus spp. biofilms grown for 10 days on glass coverslips.
Figure 2.
Laser scanning confocal microscopy images of biofilms.
(A–D (a)) Laser scanning confocal microscopy image of various grades of biofilms at × 630 total magnification. Scale bars represent 25 µM (A–D (a) Laser scanning confocal microscopy images of various grades of biofilms at × 630 total magnifications plus × 4 zoom. Scale bars represent 7.5 µM (C (b)) Image of cobwebbing. (D (b)) Image of the honeycombed region. The arrow points to the cavities in the honeycombs. Grade II biofilms presented in Figure 2 A (a and b) demonstrated few microcolonies, some of which extended to form towers. A grade III biofilm was characterised by a carpet-pile appearance with some microcolonies extending to form towers (Figure 2B (a)). Grade IV biofilms had a honeycomb appearance where the continuous layers of cells covered the coverslip with clusters forming prominences (Figure 2D (a) and (b)).
Figure 3.
Representative orthogonal view of a 10-day-old biofilm incubated under anaerobic conditions.
Laser scanning confocal microscopy image of a mature Grade IV biofilm at × 630 total magnifications plus × 4 zoom. The orthogonal view allows the representation of the 3D biofilm to be presented in 2D. The cross hairs indicate the area of the biofilm presented in 2D along the borders of the image. Depicted below and to the right of the main image are the yz and xz planes respectively. In this biofilm, there are channels between the microcolonies appearing in ‘gaps’ in the 2D structure and indicated by the white arrows. The hollow interconnecting areas under the towers are indicated by the black arrow.
Figure 4.
Scanning electron micrographs of 10-day-old biofilms.
Individual bacilli, identified as Lactobacillus spp. (day 5) (arrows) are covered by a light glycocalyx (4A (i)) and a control ATCC strain Bacteroides spp. (day 5) by a more mature biofilm (4A (ii)) both captured at × 10000 magnification. Figures 4B (i (day 5) and ii (day 10)) represent osmium fixed biofilms containing coccoid bacteria identified as Streptococcus spp. (by culture) (arrows) at × 15000 and × 30000 magnification. Glycocalyx is not well preserved using traditional aldehyde fixatives followed by OsO4 post-fixation. Figure 4C (i) (day 10) was fixed using OsO4, which stabilises lipids and figure 4C (ii) (day 10) was fixed using an aldehyde fixative followed by OsO4 plus ruthenium red to enhance the preservation of the anionic polysaccharides in the glycocalyx.