2.4. Amoeba–Bacteria Co-Culture

This protocol allows the evaluation of food selection characteristics of free-living amoebae in live conditions in drinking water through continuous/periodic observation through fluorescence microscopy (Materials and Methods section 2.4). 

Amoeba- bacteria co-culture:

1. All the bacterial strains and amoeba strains of choice were grown individually in specific media suitable for them.
2. The bacterial and amoeba strains were washed individually with the medium in which the predation experiment would be carried out
3. The correlation between cell number (CFU) determined by optical (OD at 600) and culture method was established for each bacterial strain
4. The cell number of amoeba was determined by counting on a hemocytometer (500 µL of amoeba trophozoite suspension was placed in a microfuge tube on ice for 10 min before counting with a hemocytometer).  
5. The bacterial and amoeba strains were grown in such a way that on the day of the co-culture experiment, all the strains were in their active growth phase (log phase)
6. On the day of the co-culture experiment the bacterial suspension was prepared from freshly grown bacteria and the concentration was adjusted based on the OD at 600 data (the concentration was calculated from previous OD vs Culture method correlation data). A culture method was also used to confirm the bacterial number.
7. The amoeba and bacteria were mixed at an MOI of 1:100 for individual bacteria in a tissue culture flask, 25 cm² (For example, for three bacterial strains the final MOI would be 1:300)
8. The number of amoeba trophozoites was 1- 4×10⁵ per 25 cm² flask depending on the amoeba strains
9. The flask was immediately placed on a microscope stage set at 25 °C to incubate at stationary phase (also can be done at room temperature if incubated in an incubator) and observed continuously/periodically under different fluorescence channels.
10.  Images under individual fluorescence channels and bright-field were taken after periodic intervals and the numbers of total trophozoites and with internalized bacteria (different stains) were counted from the images.
11. The number of internalized bacteria after periodic intervals was also counted in parallel co-culture experiments with the culture method.

All other details of the procedure have been provided in the following article.

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How to cite：

Shaheen, M., & Ashbolt, N. J. (2021). Differential bacterial predation by free-living amoebae may result in blooms of Legionella in drinking water systems. Microorganisms, 9(1), 174.