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We are attempting to extract bacteriophages from environmental samples. We recently had an issue with an unknown contaminant when trying to grow a lawn of M abscessus for a bacteriophage isolation spot test.

Briefly, we mixed molten top agar with a saturated M.abscessus culture and poured it over a prefilled LB plate. Then we split the plate into nine squares and placed a single drop of environmental sample filtrate in the square—the environmental samples were filtrated by a .22um PES filter.

The result looked like this:

enter image description here

As you can see, a lawn of what appears to be M.abscessus is forming (the yellow-white smaller colonies). But there are also eight circular white colonies of unknown origin.

Here is what we know:

  • The unknown contaminants are dome-shaped and look fuzzy in structure. They are roughly 5mm across.
  • They seem randomly distributed across multiple squares, which likely means the contaminant did not come from the environmental sample filtrate drops.
  • The contaminant could have been in the M.abscessus culture. But we did add Carbenicillin (100ug/ml final concentration) and Cycloheximide (1ug/ml final concentration) to the culture to inhibit bacteria and fungi.
  • The contaminant could have come from the top agar, which was stored in a 55°C water bath.
  • We created four of these plates at the same time, and they all had the same contaminant.

What species could these dome-shaped white colonies belong to?

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    $\begingroup$ Did you pour control plates without M. abscessus and without filtrate? That would tell you exactly where the contamination is coming from. As for identification, it's going to be pretty difficult without microscopic images of the colonies and without biochemical tests or sequencing. $\endgroup$
    – MattDMo
    Dec 15, 2022 at 16:56
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    $\begingroup$ From a practical laboratory standpoint, it seems like performing the sorts of controls that MattDMo suggests is going to be far more useful to you than identifying the specific contaminant. Assuming all you want to do is grow your organism free of others, it doesn't matter what is contaminating but rather what step in your process introduced contamination. $\endgroup$
    – Bryan Krause
    Dec 15, 2022 at 17:12
  • $\begingroup$ @MattDMo that makes sense. We will definitely use more negative controls on the future. We discovered that our final Cycloheximide concentration was accidentally too low, so our best guess is that this is because of that. $\endgroup$ Dec 17, 2022 at 14:06
  • $\begingroup$ Did you use a spreader and sterilise it? If you cold sterilise in pure ethanol you can pickup bugs from the alcohol.; which won’t occur with 70% ethanol. The contaminant I saw for this was Staphylococcus. $\endgroup$ Apr 3, 2023 at 5:29

1 Answer 1

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If you want to identify this bacterium or any other contaminant, you need a PCR of the 16S rRNA gene, sequence it using Sanger and then blast the results against a database. This is the easiest, cheapest, and most reliable way to know exactly what you are dealing with.

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