Week of 12/09/24

 

Introduction

Last week of lab for the year of 2024. Everything is winding down. Time to make more freeze backs and toss out expired media. We are also going to give presentations on our projects and where we wish to go from here. 

I helped out another group with their RNA isolation this week. They are attempting to get an RNA sequence of Deserti bacterial cells that have been desiccated. The process requires meticulous planning and can easily go awry. 


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 RNA isolation did not yield a sufficient amount of RNA for sequencing. 


Discussion

To increase the amount of RNA obtained, more cells are initially going to be used for desiccation. 


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