Week of 01/21/25 - Barbie Plates

 


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 microliters of chemoattractant media into one of the insertion sites

a. Use 1% agar media

11) Incubate Plates

12) Regularly Picture


Stock Solutions

Fumaric Acid

    230mg placed into 10ml of water for a 200mM solution.

Malic Acid

    270mg placed into 10ml of water for a 200mM solution. 


Results

    Two pink plates have been inoculated with P81 and two pink plates have been inoculated with D. radiodurans. Results are pending.

    The skim milk plates came out okay and we even bored a single hole into each of the plates closer to the insertion site so that the cells can immediately twitch in the direction of the attractants.


Discussion

    Fumaric acid and malic acid are going to be the attractants tested in this run. The initial insertion site will now have multiple attractants in its path. A small bored hole immediately next to the insertion site and a larger hole further along to ensure that there is a concentration gradient that keeps cells twitching further along the plate. 

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