Week of 02/10/25 Resazurin Results

 

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 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

        The SMA plates from previous weeks showed very little twitching motion. 

        The resazurin plates show a clearance zone around the center and the blue dye of the resazurin appears to dissipate within this zone. Additionally, a streak inoculated along the outside shows a lack of pink fluorescence everywhere except along the side of the plate. 

Discussion

        The SMA plates did not twitch as expected. We altered the inoculation procedure by using a broth inoculation rather than placing a colony. The lower amount of cells may have resulted in the plates drying out before the cells were able to twitch. To counter this, we are going to spin the tubes and use the pellet for inoculations.

        To better allow for directed twitch, we are going to create a concentration gradient using 1mM, 2mM and 10mM in succession to keep moving cells along the gradient.

    The plates turned clear and may simply lack Resazurin in sufficient quantities. Thus, I am going to increase the resazurin concentration from 0.45mg/ml to perhaps 10x that amount. This should keep the plates sufficiently blue before the cells twitch through it.

    To run different experiments with different gradients, attractants and dyes, I am going to request a lot of small (1 inch diameter) plates for prototypes.  We are going to test the differences between resazurin and amino black dye using these methods. 

     Resazurin works by turning from blue to pink to clear over time. To better track these transitions, it may be necessary to watch these plates on an hourly rather than a daily basis.