Step 1: Bacteria Preparation

To start our experiment, we should create an optimal growth medium, which is enriched nutrient agar for our bacteria, Vibrio anguillarium. This optimal temperature for the medium is 25ºC.

To avoid precipitation and to ensure aeration we place our bacteria in a “shaker” which optimizes the growth of the bacteria.

After the bacteria have grown, they are recovered from the erlenmeyer and put in a spectrophotometer cuvette. After that they are put in OD 600 for concentration measurement. We measure the concentration to be able to determine how much we should dilute the culture.

The bacteria is recovered from the erlenmeyer and placed in a falcon tube, then enriched agar is poured on it. We assemble them in a falcon tube to be able to reach the desired concentration which is imperative for the experiment.

To avoid precipitation and to ensure aeration we place our bacteria in a “shaker” again.

As we recover our bacteria from the shaker, we place them in microphuge tubes for further use.

To maintain the young and reproducible state of bacteria, we place them in the deep freezer which is at -80ºC.

We have a stock of bacteria which we will use later on to prepare different petri dishes to carry out the experiments.

Now that the diluted cultures are ready, we pipette them on the petri dishes, getting them ready for further experiments.

Step 2: Bacteriophage and Culture Preparation

We continue by preparing the necessary cultures with our bacteria stock and other materials. We use 6 different petri dishes. Now it’s time for the experiment.

Culture 1: Control
This culture contains only the nutrient and the bacteria (V. angullarium) population. This culture is a reference for the others so that we can determine the efficiency of each culture mix.

Culture 2:
We put antibiotics in the second culture to see its efficiency against V. anguillarum.

Culture 3:
We put the bacteria and its respective phage, Kpv-40, in this petri.
Phages are grown with vibrio anguillarum in soft agarose (LB broth supplemented with 0.3% agarose) overlays.
Once confluent, overlays were incubated with STE buffer (100 mM NaCl, 10 mM Tris-HCl buffer pH 7.4 and 1 mM EDTA) overnight at 4 °C with gentle rocking to elute the phage.

The STE-phage solution was clarified by centrifugation, sterile-filtered and incubated with 1 × PEG (4% PEG 8000, 0.5 M NaCl) at 4 °C for 1–3 days, to allow for phage precipitation.

Phages were collected by centrifugation at 10,000 g for 15 min at 4 °C and the phage pellet re-suspended in STE buffer.

We place our bacteriophage in the third culture to observe its efficiency against V. anguillarum.

Culture 4:
This culture has the antibiotic, KPV-40 and bacteria. We already mentioned the phage prep. process.
This culture is for deciding if KVP-40 and the antibiotic have synergetic effects.

Culture 5: The Synergy Culture
This culture has the bacteria, KVP-40 and an AMP.

Culture 6:
Sixth culture will only have pleurocidin and the bacteria
This is to observe the effect of the AMP alone in comparison with the synergetic effect of AMP and KVP-40

Step 3: Observations and Measurements

We have to calculate the CFU’s and PFU’s to determine whether there is a synergy between AMPs and the bacteriophage against Vibrio Anguillarum.

Overnight cultures originating from single colonies of V. anguillarum were diluted back to an OD600 of 0.05 in 50ml LB grown at 37 °C with aeration. After 15 minutes, phages were added to each culture.

At the indicated time-points, samples were collected to measure the CFU per ml and the PFU per ml.