Week of 11/18/24

 

Introduction

The plans for twitch and morph were put into effect this week. We studied the effects of proteases upon the connected P81 cells while simultaneously studying the effects of chemoattracts upon twitching P81 cells. 

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

Inoculated 8 flasks. 4 TGYB and 4 R2B. 1,2,3,4 shake and 5,6,7,8 still. Proteinase K was added to 1,3,5,7 at 10ug/ml. 1,2,5,6 TGYB. 3,4,7,8 R2b.

1. Partial rods partial alignment

2. Strings

3. Distinct rods

4. Partial alignment

5. Strings

6. Strings

7. Short rods and medium strings

8. Short rods and medium strings

Discussion

The still broths saw no variation in strings vs rods after the introduction of proteinase K. The shaken broths had minor variation in the TGY flasks. No overall discernable effect.