Week of 11/25/24

 

Introduction

The use of proteinase K has been inconclusive thus far and so we have decided to increase the concentration to 20ug/ml. Proteinase K is optimized at 50-65 degrees Celsius so its activity is slowed at the incubation temperature of P81. Hopefully increasing the concentration will account for the decrease in temperature. 

Additionally, lab is closing soon for winter break and winding down protocols are in effect. Media production has been halted to as needed only and freeze backs of samples are being created. 


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

Cells failed to dissociate even with the increased Pro K concentration. They do occasionally appear crisper.


Discussion

The Pro K and Triss combination might be destroying pili on the exterior of cells accounting for the crisper appearance. However, the concentration of Pro K might still be too low to prevent the connections of these cells from end to end from taking place.


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