Week of 11/04/24

 

Introduction

The twitch and morph assays were the assays performed this week.  The twitch assay looked at different forms of media to see if there were any differences in twitch based upon media type (in this case, pure tryptone vs pure yeast vs pure dextrose).

For the morph run, we compared TGY and R2B again to try to get consistent results with last week. We are trying to confirm that media type (more so than environmental condition) is responsible for the morphological change in which P81 rods begin to connect end to end.

Furthermore, a morph run in which we begin to apply Proteinase K took place this week. This enzyme can break down the pili between cells that forms biofilms. This week we began to calculate proportions for our stock solution. 

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 twitch plates were inoculated.

The morph run was gram stained. TGYB for samples 1-4. TGYA for sample 5. R2B for samples 6-8. 1,2,7,8 shaken. 3,4,6 kept still.

1. Strings

2. Strings

3. Rod

4. Rod

5. Rod

6. String

7. Rod

8. Rod


Discussion

For some reason, TGY broths string out most consistently in shaking conditions while R2B broths string out more easily in still conditions. Further experimentation needed to determine if there is indeed a pattern or if the results are random. 


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