Week of 02/03/25 Purple Plates


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 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)


Results

The plates have been poured and have been inoculated with P81 and D. radiodurans.


Discussion

We initially wanted to use a stock solution of 150mg/ml but this was too saturated and didn't homogenize well. We eventually found that 50mg/ml homogenizes pretty well yet I would like to lower the concentration even further in the future because a lot of residue was left behind in the 0.22um filter. I either have to use a larger filter or saturate it to a much larger degree. 

    I also wanted to use a final resazurin concentration of 0.15mg/ml but the color was not as dark blue as I would have liked when mixed with the white SMA. To account for this, I tripled the amount to 0.45mg/ml. Now I am not sure if it was too pale because it was mixed with a white media or if my stock solution simply is much less concentrated than I believe because of the resazurin left behind in the syringe (the residue that didn't make it through the filter). 

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