Week of 11/11/24

 

Introduction

This week we looked into proteases for the morphology assay. Proteinase K, Trypsin, Pepsin and carvacrol are often used to break down biofilms through different methods. Proteinase K at 10 ug/ml has been used for other species and so we will apply it here. The idea is that if the connection between cells is facilitated purely by pili connections, the dissolution of strings to rods after the application of Proteinase K will provide support for this theory. However, if the connections between cells are the result of incomplete cell division, then no separation should take place after the introduction of Pro K. 

For the twitch runs, we are introducing the chemoattractants: fumaric and Malic acid. Chemoattractants bind to twitch capable cells and ignite a pathway that results in cells twitching in the direction of the chemical gradient of attractants. Thus, if the cells are indeed twitching, they will be compelled to twitch in the direction of the attractant to a greater extent. 

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

The stock concentrations of fumaric and malic acid were created this week. We need a 2mM concentration of each. Given their respective molar masses, we needed to measure out 27mg/ 10ml and 23mg/ 10ml of malic and fumaric to create a 10x stock solution. 

Discussion

This week was focused upon preparation and research as we shift focus with the morph and twitch assays. The morph assay is going to become more protease focused while the twitch assay becomes more attractant focused.