Week of 01/27/25 Twitch is Back in Motion



Introduction

    This week we are going to introduce attractants to cells sooner in order to get a better directed motion. This should help the inoculates encounter the attractants at a much earlier stage. P81 will be inoculated as the experimental condition with D. radiodurans (a non twitcher) used for comparison. 

    Additionally, we will have to repour the resazurin plates. Autoclaving them caused them to oxidize more than we would have hoped. According to literature, if we keep the media below 50C it should remain unoxidized (which can be visually confirmed by a blue color).


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)

    -10ul of tgy broth with P81 at an OD level of 1.4

    -10 ul of tgy broth with D. radiodurans at an OD level of 1.2

10) Dispense 100-150 microliters of chemoattractant media into one of the insertion sites

a. Use 0.5% agar media

11) Incubate Plates

12) Regularly Picture


Additionally, chemo attractants were added to the media both directly next to the inoculation as well as at the far end of the plate. To accomplish this, we bored a hole into the media and filled the hole with the SMA and attractant solutions.


Fumaric acid and malic acid were used as attractants. Both diluted down to 2mM in the plate. 


Results

Immediate growth around the rim of the inoculation site. Early signs show enzymatic activity.


Discussion

    In the future, we will use 5ul for the inoculations so that a single bead of media will form at the inoculation site. This will help prevent the cells from touching the sides of the agar and growing up and over the top. To ensure that the cells are still sufficient for fast results, we will concentrate the cell solutions to an OD level of three (by taking the cells at an OD of 1 and mixing with less broth).


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