Week of 10/28/24

 

Introduction

For the morph assay, gram stains are used to identify whether the P81 rods are taking the form of single rods or connected rods. Different conditions such as shaking or stirred or TGY vs R2B are used to determine if nutrient density or environmental stability effects the ability of these rods to connect. As an aside, with the amount of gram stains necessary for the morph assay, it is wiser to place several samples (up to four I’ve found) upon a single slide to save time and materials. 

For the twitch assay, we discussed the possibility of using different dyes to be able to better visualize the twitching cells. Dyes discussed include ONBG and XGAL.

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

From R2A Samples 1-10. From TGY samples 11-20. Inoculated in R2B samples 1-4 and 11-14. Inoculated in TGYB samples 6-9 and 16-19. Sample 5 and 15 plated onto R2A. Samples 10 and 20 plated onto TGYA. Shaken: 1,2,6,7,11,12,16,17. Still: 3,4,8,9,13,14,18,19.

1. Pink rods – aging

2. Pink rods – small

3. Pink rods – small

4. Pink rods – sort of aligning

5. Purple rods – not enough ethanol

6. Pink strings

7. Pink rods and strings

8. Prink Strings aging

9. Pink strings

10. Pink rods

11. Small pink rods

12. Tiny pink rods

13. Small pink rods aging

14. Rods aligning

15. Small pink rods aged

16. Pink strings aged

17. Long pink purple strings

18. contamination

19. Pink and purple rods aligning

20. Pink rods

Discussion

The mother plate of R2A vs TGY seemed to have no effect on strings vs rods. Each R2A and TGY plate produced rods. Shaking vs still had a very limited effect. Shaking seemed to produce better strings in TGY and still produced more alignment in R2B but the biggest differences were produced by varying the media. R2B produced rods almost exclusively while TGY consistently produced strings. 

TGY vs R2B has a greater effect upon the phenotype than shaking vs still.


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