We are screening for bacteria capable of producing extracellular protease ( / proteinase / peptidase). We focus on Lactobacillaceae like Lactobacillus helveticus, Lacticaseibacillus paracasei, Lentilactobacillus parabuchneri etc. We use agar plates with UHT skimmed milk. We insert the pippette tip with bacterial suspension into the agar plate and eject the pipette.
After a few days of incubation, we indeed see the clerance zones, but, immediately around the injection site, there is a white zone first and the cleared halo is on the edge of it. See the photo.
What is the white zone ? Is seems to be located inside the agar layer, neither on the surface nor at the bottom of the agar plate. Is it the biomass of lactobacilli growing inside the agar ? Or some casein metabolite / precipitate ? Or something else ?
Let's see if I understand this picture. The hole in the center of each circle is where bacteria were injected. The tan area outside the circles is the agar containing skim milk. The dark circular area surrounding the central hole is the bacterial colony. The light ring around the dark circle is a cleared area in the milk due to the expression by the bacteria of one or more enzymes that degrade the milk protein. (The milk proteins are presumably responsible for the turbidity of the agar.) This zone of milk degradation presumably extends throughout the bacterial colony, but this can't be observed because of the opacity of the bacteria.
Another feature in the picture is that the dark circle has two parts: a central darker part surrounded by a lighter part. Perhaps it represents bacteria that have died or entered an inert state due to consumption of all the nutrients at the center of the colony.
Adam B Shapiro , yes the dark circle has two parts. The inner center is the microbial growth which can be seen on the top of the plate and also on the bottom. It looks like a normal bacterial growth I know from e.g. E. coli. What I wonder about is the extended white circle (which is dark when photographed against the window). It looks like something in the medium. I know that lactobacilli are partially anaerobic - do they grow inside the agar like this ?
I have seen this article, where opaque zone around the colony is interpreted as weak proteolysis, and clear zone means strong proteolysis. Article Assessment of Probiotic Properties and Characterization of S...
I would still wellcome some more information. It seems like I have opaque zone surrounded by a clear halo. Why would I have weak proteolysis near the colony and strong proteolysis further away ?
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