Week of 10/14/24


 Introduction

We did another run of the morph project. It was difficult to obtain high quality gram stains last week so we are adjusting our approach and increasing staining efficiency. 

The twitch projects are ongoing. It takes approximately 10-14 days to get results with this assay.

Meanwhile Nick and Riyah are working on their UV projects and Andrew is working on his Salt survival project. 


Methods

Morph project – From both an R2A plate and a TGY plate: two TGY plates, two R2A plates, two R2B broths and two TGY broths were inoculated. 24 hours later these sources were gram stained.

Twitch project – Sugar water attractants. 1 gram of dextrose per 1000ml. This solution was poured into an indentation within the media plate to be used as a chemoattractant.

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

Strings within broths and rods within plate cultures as observed via gram stains. Several of the sources were unfortunately contaminated with staph.


Discussion

Whenever possible, use the hood room to inoculate to avoid the potential for staph contamination. 

The sugar water attractant concoction might have been ill-advised. First, there is already sugar within the skim milk media and thus dextrose might not work the best as an attractant. Secondly, using a liquid and then waiting for it to dry might not allow it to adequately diffuse throughout the media. A different form of chemoattractant is necessary and perhaps a different delivery method as well. 


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