For some time I have been under the suspicion that my bacterial cells are undergoing "autolysis". The cells are grown in minimal media with at least 1% carbohydrate supplement. It is worth mentioning that these cells (Geobacillus thermoglucosidasius) can sporulate.
I'm looking for an efficient protocol to detect and perhaps quantify nucleic acid in bacterial culture supernatant. Does anyone have suggestions? Thank you!
Did you try detecting the nucleic acids using a spectrophotometer? Also a Nanodrop will allow you to quantify the nucleic acids. You can also try using an assay to detect the presence or absence of nucleic acids in the samples.
Maybe first make sure you filter out everything including spores.
You can then precipitate any nucleic acid there might be with isopropanol. (If you volume is larger than 2-3 ml, I would first evaporate it down to this volume).
Proceed normally as you would when extracting DNA/RNA, that means wash with EtOH and resuspend the nucleic acid pellet in H2O.
Then quantify on NanoDrop and run on a 1.8% Agarose Gel.
If you have mainly RNA, you will see distinct rRNA bands, if its DNA you will see a smear.
Maybe you could also try picogreen (Molecular Probes). This fluorescent dye specifically binds to dsDNA and detects it even at very low concentrations. I can imagine that it should work also for not purified dsDNA in culture medium, but I’ve never tried it.
You have to extract the DNA/RNA from the supernatant in order to quantify it. There are commercial kits available to do this, thereafter you can use a Nanodrop or Spectrophotometer to quantify the DNA or RNA.
Mere detection of DNA in supernatant can be tested by fluorescence under UV light source. Alternatively a controll of the test broth and suspecetd DNA sample be kept and absorption at 260nm recorded as DNA absorbs maximally at 260nm. Alternatively DNA can be quantified by diphenylamine reagent method by intensity of blue color.
Have you tested for DNAse presence in your media? Simple colormetric analysis required. Confirm your suspicion. What temperature are you incubating G. thermoglucosidasuis at.? I will assume you have found an optimum temperature for avoiding sporulation altogether. Devil in the details....
For the sake of argument and the continuity of this exchange I have I few points I’ve ponder over some time. Thank you all for the feedback, I truly appreciated. Plus is fun!
Reyghana - The answer to your question is yes I have try spectrophotometry, however the readouts are not exactly reliable. I’m under the impression the media may be of enough complexity to throw spectrophotometer readings off from sample to sample. Like you mentioned in your second response I have to extract the nucleic acid. I’m currently trying such approach for the sake of assessing its effectiveness. I do appreciate your feedback.
Ulrike - DNA precipitation was my original though however I have a significant number of samples that may create a complex process altogether. I would leave this option low in priorities while searching for a good way to do approach this expeditiously.
Kasia – Picogreen seems like a sensitive solution for this challenge. I read on this and the specificity and efficiency appears to be great. In terms of cost effectiveness we’ll have to “bite the bullet” considering the amount of samples I’ll be generating but if the data is good then it may be worth the investment. You mentioned you never try culture detection, do you have experience with this particular method in similar scenarios. If so, what are your thoughts on its performance?
Nicholas – I got a previous mention of picogreen in the suggestions. Like I a ask her I’ll ask you; Must one need to absolutely purify nucleic acid or the technique allows for less than clean medium? Have you ever implemented the technique on similar conditions?
Kapil – I agree that theoretically one can detect DNA in medium via UV detection. However, an indicator must be used. Something like ethidium bromide intercalation I presume. Have you thought of anything else? I do like the diphenylamine reagent approach I will explore this particular technique. Thank you.
David Hayes – I agree with your assessment that perhaps a colorimetric assay will do the trick. G. thermoglucosidasius ranges from 52 – 70 degrees. Optimal growth temperature is 60 degrees. Taking that as a middle point we have explore both side of the range but sporulation, we suspect, has more to do with starvation since we grow it in minimal medium. This can also be observed in rich media after several days when the batch culture has reach a near depletion state. Haha! Devil in the detail... indeed. You sound like you work with a similar bug? Thanks for the feedback.
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