S-STEM Scholar Evan Russell's Blog

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 ...

 Introduction We are going to attempt the Pro K morph assay again. This time we are using 40ug/ml which is four times the amount used in the original protocol. Hopefully the increased concentration will increase our chances of cell dissociation.  Additionally we are going to look at twitch plates that have had attractants added in one quadrant with the hope that it will show directed twitching.  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 and 0.5% agar) 2) Prepare a sterile hood by wiping everyth...

  Introduction The use of proteinase K has been inconclusive thus far and so we have decided to increase the concentration to 20ug/ml. Proteinase K is optimized at 50-65 degrees Celsius so its activity is slowed at the incubation temperature of P81. Hopefully increasing the concentration will account for the decrease in temperature.  Additionally, lab is closing soon for winter break and winding down protocols are in effect. Media production has been halted to as needed only and freeze backs of samples are being created.  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 express...

  Introduction The plans for twitch and morph were put into effect this week. We studied the effects of proteases upon the connected P81 cells while simultaneously studying the effects of chemoattracts upon twitching P81 cells.  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 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 co...

  Introduction This week we looked into proteases for the morphology assay. Proteinase K, Trypsin, Pepsin and carvacrol are often used to break down biofilms through different methods. Proteinase K at 10 ug/ml has been used for other species and so we will apply it here. The idea is that if the connection between cells is facilitated purely by pili connections, the dissolution of strings to rods after the application of Proteinase K will provide support for this theory. However, if the connections between cells are the result of incomplete cell division, then no separation should take place after the introduction of Pro K.  For the twitch runs, we are introducing the chemoattractants: fumaric and Malic acid. Chemoattractants bind to twitch capable cells and ignite a pathway that results in cells twitching in the direction of the chemical gradient of attractants. Thus, if the cells are indeed twitching, they will be compelled to twitch in the direction of the attractant to ...

  Introduction The twitch and morph assays were the assays performed this week.  The twitch assay looked at different forms of media to see if there were any differences in twitch based upon media type (in this case, pure tryptone vs pure yeast vs pure dextrose). For the morph run, we compared TGY and R2B again to try to get consistent results with last week. We are trying to confirm that media type (more so than environmental condition) is responsible for the morphological change in which P81 rods begin to connect end to end. Furthermore, a morph run in which we begin to apply Proteinase K took place this week. This enzyme can break down the pili between cells that forms biofilms. This week we began to calculate proportions for our stock solution.  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 ...