Week of 02/17/25 Problem Solving

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, almost regardless of the attractant or the repellent present, the cells seemed to twitch in a single random direction rather than in a circle or towards a pellet. We are looking into possible causes for this as well. 

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 microliters of chemoattractant media into one of the insertion sites

a. Use 1% agar media

11) Incubate Plates

12) Regularly Picture

Resazurin Plates

1) Create 50mg/ml Resazurin Stock Solution in PCR water

2) Filter at least 3ml of the Resazurin Stock Solution into a 15ml conical vial using a 0.22um filter and syringe

3) Melt down media (in this case 0.75% SMA with 1% Agar)

4) Let media cool to at least 50C (verify with temp gun)

5) Pipette 9ul of stock solution for every ml of media (e.g. 900ul for 100ml of media) to create a final concentration of 0.75mg/ml

6) Stir Well

7) Pour Media into Plates (Molds optional)

New Twitch Inoculate Considerations

1) Inoculate Broth with Desired Culture

2) For each inoculate, reach an OD of 1.0

3) Place 1ml in an eppendorf tube

4) Centrifuge for 60sec 

5) Remove supernatant

6) Place Pellet at the Center of the Twitch Plate

Twitch Plate Considerations

1) Create stock of attractants at 1mM, 2mM and 10mM

2) Create three holes away from the inoculation site in twitch plate

3) Inoculate from nearest to farthest 1mM, 2mM and 10mM respectively

4) Create a concentration gradient for the cells to migrate towards

Results

        We have bored the new holes in the plates.

        We have utilized the new concentrations (1, 2, and 10mM) consecutively from the center of the plate. 

        We have a control run in the lit incubator as well as the no light incubator all set to a consistent alignment. 

        We got the inoculates to an OD of 1.0 before pelleting the cells and inoculating the plates.

        We inoculated control fumaric and malic plates to confirm that cells can grow through 2mM concentrations of these chemicals. 

Discussion

       To increase the effectiveness of resazurin, we are going to try multiple media types with an increased concentration. We are going to mix TGY, TSB and R2A medias of (with 10, 50 and 100% concentrations each) with a resazurin concentration finalized at 4.5mg/ml vs the previous 0.45 mg/ml. Additionally we are going to track these plates more closely over time to account for rapid changes in color from blue to pink to clear. 

        The cells have not been cooperating as far as the direction that they choose to twitch in. To encourage the cells to twitch in a controlled direction, I am going to control for certain unexpected variables. The cells in the incubator appear to all twitch in the same cardinal direction regardless of their environment. I am going to begin aligning the plates so that outside forces like light and heat can't influence the direction of twitch. It is also a concern that the pellet near the inoculation sight may be too dense and thus unable to allow for twitching motion. To counter this, I will create three extra pellets surrounding the inoculation site to act as controls. To further increase our capacity to direct twitch, we are going to create a concentration gradient of three pellets consisting of 1, 2 and 10mM concentrations of fumaric and malic acid concentrations. The cells should in theory move up the concentration gradient as long as these chemicals are functioning as attractants.

        Malic acid is apparently functioning as a repellent. To confirm this, we are going to grow the bacteria on control plates set at 2mM concentrations. If they can grow than the Malic is truly repelling rather than simply inhibiting growth.

        To increase the overall amount of twitch that can occur, we are going to increase the inoculate by taking a broth sample (standardized to an OD of 1) and pelleting the sample and then using the pellet as an inoculate. 

        Finally, to get a wide range of species data, we are going to take all of the different data that we have gathered to finalize a procedure with the perfectly crafted media to grow 8 to 10 species and compare their twitching capacity.