Week of 10/07/24

 

Introduction

The twitch project looks at the twitching motion of Deinococcus aquaticus P81 cells as they spread along a plate of media in response to metabolites in the environment. Using minimal media may help prompt cells to twitch to a greater extent as they spread in search of nutrients. 

The morphology project looks at the elongation of P81 when they are grown in broths instead of upon a plate. The current theory is that the cells are connecting end to end via pilli connections. We are looking into environmental and media conditions that may induce a morphological change. We are currently comparing shaking vs still conditions as well as TGY vs R2B broth. 

Methods

Morph project - From a stock TGY plate we inoculated a R2A plate, TGY plate, two R2B broths and two TGY broths. One of each of the broths was placed in a shaking incubator while the other was placed in a still incubator.

Twitch project – we inoculated skim milk plates of 0.5% skim milk and 0.75% skim milk concentrations. P81, D. pimensis, D radiodurans and D. xinjiangensis were inoculated upon 0.5% and 0.75% plates in duplicate.

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

We looked at the SMA twitch plates from 09/27/24. We were able to observe a large twitching presentation from P81. D radiodurans didn’t present any form of twitch but did show a large clearance field around the inoculation site. D xinjiangensis and D Ficus both showed slight twitch and a slight clearance field. 

For the morph project, we gram stained the plates and broths and observed elongation in the broths and no elongation upon the plates.

Discussion

P81 appears to exhibit the greatest degree of twitching motion while D. radiodurans showed the least degree of twitching. However, the degree of enzyme activity appears to be inversely proportional to the degree of twitching as P81 showed the least amount and D. radiodurans showed the greatest amount of enzymatic activity. Ficus and xin both appeared to exhibit medium amounts of enzymatic activity and twitching motility.

For the morph project, we confirmed the formation of elongated cells when grown in a broth. Interestingly enough, the TGY broth that is shaken and the R2B broth that is kept still both appear to create the greatest amount of elongation. Further research will be conducted to ascertain why this different environmental condition might result in the elongation phenomenon.