Some minor setbacks

Hello passengers, this is your captain speaking. We are experiencing some  minor turbulence, please fasten your seatbelts and remain seated.  All attempts at being funny aside, the project assessing the duration of a certain bacteriophage’s phase of infectivity is experiencing some tiny (but annoying) setbacks. First of all, let me bring you up to speed.

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After completing the second round of experiments to determine which extraction method would be best to use in this experiment, I finally chose blending as the extraction method. I then set up three sterile microcosms, all filled with the same autoclaved soil. Two of the microcosms were inoculated with T4 while the last microcosm had autoclaved water added. From these three microcosms, three separate extractions were carried out with blending and a buffer. Said extractions were plated to determine pfu and mounted onto an anodisc slide (with replicates). Upon examination of the slides, it was discovered that the control slides exhibited some kind of fluorescence which was not the correct morphology to possibly be T4 particles. The microcosm extractions, plates, and slides were repeated twenty-hours and four days after  the first extraction. More than a hundred pictures and many many counts later, it is heart retching (soul crushing may be more appropriate here) to still have these unexplained DNA or RNA containg hooligans (which look like non-T4 viruses) to be dispersed throughout your control anodiscs.  I happened to set up another control microcosm using SM buffer instead of water and the results are promising but not good enough.

As such I will be conducting an experiment in which I test water and SM buffer microcosms with the blending extractions and all autoclaved material. In addition, one of the replicates from each microcosm will be treated with DNase. If there is any free-floating DNA or RNA , or  anything capable of picking up the stain and not encased , in the microcosms the DNase should chop it up. I am conducting this mini-experiment in hopes that I can determine a way to setup accurate microcosms which will lead to accurate counts. Wish me luck !

Until next time,

Krysten

Comments

  1. Yup, that’s how research goes. I’m getting fairly stuck myself as well! Hang in there!

  2. dmhardbower says:

    I’m sorry, Krysten!! The microcosms were a lot of trouble last summer too!! Have you considered that possibly autoclaving isn’t enough to remove all viruses from the soil? Maybe the ability of some viruses to adsorb to the soil particles is protecting them from the heat and pressure somehow? Were the virus particles that were not T4 present on the experimental slides as well and not just the control slides? Just a thought! You’ll get there!!