Listeria

Because Constructive Microbes constitute a mixture of living cells of a number of bacterial species, in the first step of the experiment the liquid (diluted 1:5 and 1:10) was applied on the PALCAM medium. The medium was incubated aerobically at 30oC for 48 hours. None of the microorganisms included in Constructive Microbes was able to grow on the medium. Thus, the PALCAM medium could be used in the further steps of the experiment and any evidence of aesculin-positive colonies would result exclusively from the presence of living cells of Listeria monocytogenes added to the liquid.

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The suspension of L. monocytogenes prepared as described previously (0.5 of the McFarland standard) was mixed with the undiluted Constructive Microbes liquid at the 1:1 ratio (2ml of Listeria suspension + 2ml of liquid) and allowed to interact at room temperature. After 24 and 48 hours, as well as 5 days of the test, 0.5 ml of the mixture were diluted 1:10, 1:100, 1:1,000 and 1:10,000 with saline and 0.2 ml of each dilution were inoculated onto PALCAM medium.

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The plates were then incubated at 30oC for 24 hours. At each time assessed, the number of living cells of L. monocytogenes was calculated based on numbers of aesculin-positive colonies grown on the medium. The number of viable cells was not decreased, as compared with the initial value, even after 5 days of contact of Listeria with the Constructive Microbes liquid. However, it was noticed that the intensity of Listeria growth (expressed as the diameter of colonies grown) was strictly dependent on the concentration of Constructive Microbes. The preparation diluted 1:10 fully inhibited the growth of L. monocytogenes (no visible colonies, Fig. 1). As the dilution of the mixture of Constructive Microbes and Listeria cells increased, the obtained bacterial colonies were larger and larger (ca. 0.1-0.2 mm at 1:100 dilution [Fig. 2], approx. 0.5 mm at 1:1,000 and approx. 1 mm at 1:10,000 [Fig. 3]).