Crystal Violet and Twitch Motility Assays

Introduction:

This blog is both for the full week Nov 18-22 as well as the three days before thanksgiving break. We were able to do a full pre-desiccation to RNA isolation procedure as well as help with a number of various procedures in the lab. This Blog is covering the other procedures done this week in lab. 



We were  able to get our working and permanent freeze backs from the A, B, C, D and E plates inoculated last week. On the twenty-second, after the RNA isolation, five flasks were inoculated from each of the working freeze backs. After growing over the weekend, we noticed a difference from the normal growth. 

Usually the flasks end with a small amount of growth in the media and a large ball/clump of cells gathered. This was thought to be due to the movement, however others notated that in all the previous semesters when they were working with deserti, it grew homogenously with none of the clumps we have been getting that have continuously worsened over the semester. When we started working with it the growth in flasks had very tiny clumps throughout the flask that have gradually turned into the large single clumps we see in flasks lately, with seemingly most of the growth within 48 hours grouped together. 

But these 5 flasks had little to no clumping, with one flask having a single small clump and one having a few tiny clumps with the rest being homogenized, and the other three with no clumps at all. Because of this the five flasks were streaked onto plates and we will see if we can replicate this next week. The clumping may relate to the gradually smaller/rounder shape we have been seeing during gram stains, and the worsening RNA isolations. 

 


To start with the assays, we were able to assist Logan with Crystal Violet assay. We inoculated three rows of a 96 well plate with Deinococcus sonorensis and six rows of a second 96 well plate with Deinococcus xinjangensis.

General Biofilm Assay Protocol

Materials Needed:

1 round-bottom 96-well plate

1 flat-bottom 96-well plate

Appropriate media

Appropriate antibiotic stock

15 mL conical tubes or glass test tubes for growing liquid cultures

0.1% crystal violet

30% acetic acid

Paper towels

A large beaker

A tray or box that is slightly larger than a 96-well plate

A plate reader

1. Prepare 5mL LB liquid cultures (supplemented with appropriate antibiotics and

inducers) from stock or a colony from the transformation plate of the desired strains.

Allow the cultures to grow for 18-20 hours at 37 degrees Celsius in a shaking incubator.

3. Prepare 1:100 dilutions, with a total volume of 1 mL, with desired media for each

liquid culture.

4. Plate 100 μL of each dilution in sets of 4 wells in a round-bottom 96 well plate.

5. Cover the 96-well plate and incubate at 37oC for 48 hours.

6. Shake the plate out over a tray to remove all planktonic bacteria.

7. Rinse the 96-well plate in a large beaker of water and shake the water out over the

tray.

8. Lay a paper towel out on the bench top. Hit the 96-well plate against the covered

bench top until no liquid remains in the wells.

9. Stain all wells used in the assay with 125 μL of 0.1% crystal violet for 10 minutes.

10. Shake the 96-well plate over the tray again and rinse out the crystal violet in a large

beaker of water.

11. Cover the bench top with more paper towels and hit the plate against the bench

top until all wells are free of liquid crystal violet. Note: Make sure that the only crystal

violet remaining is bound to a biofilm at the bottom of a well. Rings of crystal violet

around a well are not indicative of biofilm formation and should be rinsed again, as

excess stain will skew the results of the assay.

12. Leave the plate face up on the bench top overnight to dry.

13. Add 200 μL of 30% acetic acid to all wells that were stained to solubilize the crystal

violet. Allow the acetic acid to sit for 10 minutes.

14. Pipette up and down the mix the acetic acid and crystal violet in the wells.

15. Transfer 125 μL of the acetic acid/crystal violet solution from each well into a well in

a flat-bottom 96-well plate.

16. Read the OD595nm of each well in the flat-bottom plate with a plate reader.

17. Subtract the average of the blank wells from the OD of each well that contained a

sample.

18. Calculate the average of the sets of wells containing the same sample.

19. Normalize the averages to the average of the control wells.  



I was also able to help Evan with a twitch motility protocol. We had 14 thin plates with wells already inside. Depending on the number of the plate we added one or both chemoattractants; Fumaric acid and Maleic acid. I was the on adding the Fumaric acid depending on the appropriate plates. The chemoattractants were in agar that we were pipetting into the premade wells, making us move quickly as it would solidify in the pipette tips if we didn't go fast enough. After the two solutions were pipetted in we waited 15 minutes for them to solidify, then inoculated the correct bacteria into the middle well in the center of the plate, again using a pipette tip. 

Through pili twitch motion the cells should twitch towards the chemoattractants overtime, visualized by a 'flowering' effect as the cells move out from the center.


Plate 1: p81 control

Plate 2: d.rad control

Plate 3: Maleic acid, p81

Plate 4: Maleic acid, p81

Plate 5: Fumaric acid, p81

Plate 6: Fumaric acid, p81

Plate 7: Maleic and Fumaric acid, p81

Plate 8: Maleic and Fumaric acid, p81

Plate 9: Maleic acid, d.rad

Plate 10: Maleic acid, d.rad

Plate 11: Fumaric acid, d.rad

Plate 12: Fumaric acid, d.rad

Plate 13: Maleic and Fumaric acid, d.rad

Plate 14: Maleic and Fumaric acid, d.rad

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