Week of 12/02/24

 Introduction

We are going to attempt the Pro K morph assay again. This time we are using 40ug/ml which is four times the amount used in the original protocol. Hopefully the increased concentration will increase our chances of cell dissociation. 

Additionally we are going to look at twitch plates that have had attractants added in one quadrant with the hope that it will show directed twitching. 

Methods

Morph Assay Protocol

1) Inoculate mother plate with sample

2) Inoculate R2A and TGY Plates and Broths

3) Incubate Plates and Broths

a. Some broths will be shaken and some kept still 

4) Gram Stain Plates and Broths after at least 24 hours

5) Record and compare results

6) If needed, introduce a protease to break apart pili connections

7) Re gram stain after 24 hours

8) If needed, run an RNA sequence to check gene expression

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) Inoculation center of plate (directly onto the plastic)

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

Results

We appear to have directed twitching.

The increased Pro K did not yield conclusive results. 

Discussion

The morphology experiment might have to be shelved for now. The year is coming to an end and we have to focus our attention on more promising leads…like the twitch project.

The twitch assay has yielded positive results, but there are complications. Visually, it is difficult to view the directed cells and so we are looking into using dyes upon the cells to be able to take high quality pictures. Additionally, the twitching appears to happen too late. There are two possible solutions to this issue. Firstly, we can let the attractants diffuse to a greater extent before we inoculate. Secondly, we can redesign the molds to help the attractants meet the inoculate sooner. 

Letting the attractants diffuse before inoculation comes with its own issues. Primarily, the plates have a short shelf life in the incubator and the twitching process itself already takes two weeks (10-14 days). Thus, it would be wiser to create new twitch plates that simply introduce the chemo attractant sooner. Ill discuss this with Micheal, our resident 3D printer operator.