Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Abstract

Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms ( Lambert et al., 2003 ). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.

Keywords: Bdellovibrio bacteriovorus; Predatory bacteria; Predatory quantification.

Figure 1.. Workflow to set up Bdellovibrio co-cultures.

A. Preparation of the prey cell suspension (here, P. putida KT2440). B. Preparation of Bdellovibrio cells. Two-step cultivation of Bdellovibrio is needed to obtain the predatory cells for the experiment. C. Double layer method to quantify the cell number of B. bacteriovorus HD100. D. Development of B. bacteriovorus HD100 on a lawn of prey on DNB agar plates after 2-3 days of incubation at 30 °C. Bdellovibrio cell number can be quantified as plaque-forming-units (pfu/ml).

Figure 2.. Predation profile to study the predatory capability of B. bacteriovorus HD100 upon P. putida KT2440.

A. Schematic representation of the co-culture involving B. bacteriovorus HD100 and the control culture of P. putida without predator. B. Viability of the prey and predator cells at the beginning of the experiment (0 h) and after 24 h of incubation. The purple bars represent the viability of the prey cells and the orange bars correspond to the predator viable cell number. Error bars indicate the standard deviation of the mean (n ≥ 3).

Figure 3.. Co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 under the microscope.

A. Co-culture at the onset of predation (time zero); B. After 24 h of incubation, only predatory cells can be observed. Cultures are routinely visualized using a 100x phase-contrast objective and images taken with a Leica DFC345 FX camera.