S-STEM Scholar Evan Russell's Blog

Introduction          We have observed that the resazurin compound appears to be cleared by enzymatic activity created by the inoculates. Alternatively, it is possible that the resazurin is turning from blue to pink to clear before we are able to observe the color shift or perhaps the concentration of resazurin is simply to low to be able to adequately see the pink color.           We have also observed that the cells do not seem to twitch to the same extent as they did last semester. This may be caused by a lower concentration of cells initially inoculated.          Malic also appears to act as a repellant as cells grow around the malic pellet. It also is possible that malic simply kills the cells and inhibits growth rather than acting as a true repellent. We also want to look into whether or not the density of the pellet itself is also acting to inhibit growth.          Oddly, almo...

  Introduction          This week we received our first look at the twitch plates mixed with resazurin to try to better visualize results. We looked at building better concentration gradients for the chemoattractants as well. Overall, we decided to investigate multiple new avenues to better track twitch using different dyes, plate sizes and chemoattractant gradients.  Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 0.5% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes containing five pronged molds to allow for insertion sites a. Use a sterile 10 ml pipette 6) Let plates cool 7) Remove molds 8) Use UV light for 15 minutes to ensure sterilization of plates  9) Inoculate center of plate (directly onto the plastic) 10) Dispense 100-150 m...

Introduction This week we are going to use SMA plates mixed with Resazurin to show the spread of our twitching bacteria in a way that is both visually pleasing and easy to spot. The resazurin mixed with SMA will create blue or violet plates that should turn pink in areas where bacteria are present because the process of reducing NAD+ reacts with the resazurin and changes the color. Thus, even when we cant see the twitching cells anywhere besides the inoculation site, we will still be able to see where they are by the color shift. Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 0.5% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes containing five pronged molds to allow for insertion sites a. Use a sterile 10 ml pipette 6) Let plates cool 7) Remove molds 8) Use UV light for 1...

Introduction     This week we are going to introduce attractants to cells sooner in order to get a better directed motion. This should help the inoculates encounter the attractants at a much earlier stage. P81 will be inoculated as the experimental condition with D. radiodurans (a non twitcher) used for comparison.      Additionally, we will have to repour the resazurin plates. Autoclaving them caused them to oxidize more than we would have hoped. According to literature, if we keep the media below 50C it should remain unoxidized (which can be visually confirmed by a blue color). Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 1% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes containing five pronged molds to allow for insertion sites a. Use a sterile 10 ml...

  Introduction     We prepared twitch media this week! Chad was kind enough to provide plates for twitch. The pink "barbie" plates should slightly change color as NADH is metabolized upon the media. Additionally I prepared some trusty 0.75% skim milk agar (at 1.0% agar). I placed molds within the media to create inoculation sites and sites in which I can place attractants.  Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 1% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes containing five pronged molds to allow for insertion sites a. Use a sterile 10 ml pipette 6) Let plates cool 7) Remove molds 8) Use UV light for 15 minutes to ensure sterilization of plates  9) Inoculation center of plate (directly onto the plastic) 10) Dispense 100-150 microliter...

Introduction     Welcome back everybody! This week we got the lab up and running again. Our main objective was to get our cells passaged from freezeback. Additionally, we began mixing and autoclaving media so that we would have plates and broths to work with. This year we decided to use slants instead of plates for stock for the added benefit of utilizing slants over the course of three months when plates would only ever keep cells fresh for maybe two weeks.      This year in the world of twitch media, we are experimenting with different dyes within the agar to better visualize the movement of cells. Updates coming next week on dyes that we decide to utilize.  Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 1% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dis...

  Introduction Last week of lab for the year of 2024. Everything is winding down. Time to make more freeze backs and toss out expired media. We are also going to give presentations on our projects and where we wish to go from here.  I helped out another group with their RNA isolation this week. They are attempting to get an RNA sequence of Deserti bacterial cells that have been desiccated. The process requires meticulous planning and can easily go awry.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene expression Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA ...