czernobill

Hello,

I used anaerobic bags to test if my bacterial isolates could grow in the absence of oxygen. There was clearly visible, hence little growth. In contrast, when incubating aerobically strong growth is observed. Thus, I would consider the bacteria as facultatively anerobic.

As negative control I used Micrococcus luteus which did not show any growth.

But: My positive control (Staphylococcus saprophyticus, facultatively anerobic) did not grow/very very little in the anaerobic bag. I am confused because I expected them to grow. I do not have a strictly anerobic bacterium. I do not have anaerobic indicator strips.

The questions I would like to ask is whether this method is adequate in judging a species as facultatively anaerobic and why S. saprophyticus did not grow. I inoculated from fresh cultures on appropriate medium.

Furthermore, does anybody know for which purpose oxygen is metabolized in anaerobic spp.? From this metabolization H2O2 is produced which cannot be detoxified. But why do they use oxygen?



Thanks a lot!

Hello,

I isolated bacteria from a natural lipid mixture which has shown to exert antimicrobial effects in the diffusion test. I am interested in whether the antimicrobial substances are of bacterial origin or if the native lipids themselves are antimicrobial.

The bacteria are lipophilic. When growing them in liquid medium supplemented with Twee-80 as lipid scource and afterwards performing a diffusion test with the liquid culture there is no antimicrobial effect.

But it might be that the bacteria use the lipids in the mixture, modify them and thereby conferring antimicrobial activity.
How can I test for that?

On the one hand, I need to include the lipid mixture in the growth medium, on the other hand I have get rid of them before conducting diffusion test to make sure that only bacterial metabolites will be included in the diffusion test.


My idea is preparing agar medium where I substitute Tween-80 for the natural lipid mixture. I inoculate the agar plates with the isloated bacteria and incubate. The colonies will be taken and suspended in water and a diffusion test will be conducted.

What do you think? I am wondering if the microbial metabolites will diffuse into the agar in that the diffusion test will lack any antimicrobial activity - even if the bacteria produced antimicrobial substances from the lipid mixture.

Thank you very much!

Hi there,

I am trying to do some tests on bacterial motility using B. subtilis and E. coli as positive controls, M. luteus as negative control and one unknown bacterium. 16S data suggests the genus Corynebacterium. Thus, it should not move.

I conducted a wet mount test with a 25h old broth culture of these organisms but could not find one single moving E. coli cell (up to 1000x magnification). Regarding, B. subtilis, few cells were observable at 400x magnification.
The unknown bacterium did not exhibit an motion, but the test isn't valid because E. coli didn't move as well.


In another test I preapared test tubes containing ordinary LB medium (0.4% agar content). I inoculated the species in question with a needle and incubated until growth was good. B. subtilis grew at the surface only. E. coli showed enhanced growth were the needle was inserted.
My unknown bacteria exhibited little growth in the upper portion of the path of the needle. Additionally, there was a considerable and diffuse turbidity right below the surface of the medium.
I would rate this as motility, but Corynebacteria are non-motile.


What can I do? Has anyone tried to include MTT (maybe 0.5%) as indicator of growth?
Are there different, easy to perform tests for motility?


Thanks a lot!

Hi,

when I am doing phlyogenetic analysis after gene seuqencing of my bacteria, I very often obtain a tree like this (rpoB gene):



Here, what I understood is that the samples make a cluster in 99% of the cases and thus belong to the same species.
The bootstrap value (500 replicates) at the node of the samples and the closest relative is only 60. Sometimes, it is even less for other trees. I am not sure about how to interprete this value.
I think it means that in 60% of the calculations the tree bifurcates into two branches giving rise to the shown topology.
I am note sure what the remaining 40% could look like. Could it have more than two branches or could the samples and species 24 fall into one cluster?
What does it mean and what can I infer from such a tree?


(The highest similarity in Blast search normally is <90% for rpoB.
Additionally, I will do biochemical tests and a DNA fingerprint using the samples and the closest relatives regarding the blast search.)


Thanks a lot!

Hi,

I isolated bacteria from a dog, sequeneced the 16S rRNA gene and aligned it (similarity after manual correction and BLAST search ~96%). Then, I sequenced two other protein coding genes yielding a homology of <90% to the closest relative. I did the whole procedure twice. A few month ago, I isolated bacteria, selected one single colony and sequenced it. Now, I isolated again and selected three colonies. I streaked them on plate several times and again sequenced the genes.

I am wondering why all the sequences from all the colonies are 100% identical. Shouldn't there be slight differences, i.e. different strains?
Will people believe that I isolated twices if the sequences are fully identical?


Thanks a lot!