Source: Wunsch, C. M., et al. Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells. J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.
The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols may be used for a number of anaerobic pathogens. Although HUVECs are used, this protocol may be used for other eukaryotic cells both immune and non-immune.
1. Anaerobic Chamber Use and Maintenance
Note: P. gingivalis is an anaerobe sensitive to normal levels of oxygen encountered in ambient air. A controlled anaerobic environment is vital for the cultivation of P. gingivalis.
2. Preparation of Anaerobic Bacteria
Note: P. gingivalis is aerotolerant and can be stored in aerobic conditions but it will not grow in the presence of oxygen at levels higher than 6%. An anaerobic chamber is necessary for the proper cultivation of P. gingivalis and other anaerobic species (Figure 1). Proper training and education on anaerobic chamber use are required before working with micro anaerobes.
Note: The optical density of the bacterial suspension is determined and the bacterial concentration for each strain to be examined is adjusted. For P. gingivalis a suspension at OD660 of 0.7 corresponds to mid-log phase and ~7 x 108 cells/ml. Growth conditions described in the protocol above are specific for P. gingivalis and may need to be adapted for other bacterial strains.
3. Endothelial Cell Culture
Note: Purchase pooled primary HUVECs and culture in basal medium containing vascular endothelial growth factors (VEGF) at 37 °C in 5% CO2 according to manufacturer's instructions.
4. Survival Assay Invasion/Interaction (Plating)
Note: When performing this assay, prepare two 6-well plates of endothelial cells seeded at 400,000 cells/well. One plate will be used to assess bacteria attached to and internalized by host cells. The other plate will account for intracellular bacteria. The 6-well plate allows for triplicates of two samples to be performed in one experiment. For an outline of this protocol please refer to the survival assay flowchart (Figure 2).
Figure 1. Anaerobic vinyl chamber and its components. (A) A vinyl anaerobic chamber sealed completely from atmospheric oxygen provides workspace for two individuals at a time (32 in x 78 in). It contains an incubator set at 37 °C (back middle). (B) An airlock is used for the transfer of items from the lab environment to the anaerobic chamber. Pictured is an automatic airlock operated through a controller that can be programmed to automatically perform the vacuum and purge procedures needed to create an anaerobic environment. (C) A Hydrogen Sulfide Removal Column provides maintenance-free high-capacity removal of undesirable hydrogen sulfide. (D) Two catalyst fan boxes are placed throughout the anaerobic chamber to help circulate the chamber's atmosphere through palladium catalyst, which, in the presence of hydrogen, removes oxygen. The anaerobic chamber is set up according to the manufacturer's instructions.
Figure 2. Schematic representation of a protocol used for survival of anaerobic bacteria with eukaryotic cells. Both assays for total bacterial survival and survival of internalized bacteria can be performed at the same time.
The authors have nothing to disclose.
Vinyl Anaerobic Chamber-Type B | Coy Laboratory Products | Model 2000 incubator | |
TSA II Trypticase Soy Agar w/5% Sheep Blood | BBL | 221261 | |
Human Umbilical Vein Endothelial Cells 10-donor Pool | LifeLine Technology | FC-0044 | |
VascuLife VEGF Medium Complete Kit | LifeLine Technology | LL-0003 | |
TrypKit | LifeLine | LL-0013 | |
Saponin | Riedel-de Haen | 16109 |