Week of 02/24/25 New Problems, Old Solutions



Introduction

    The attempt of standardizing the initial inoculate has led to too many new issues and thus must be discarded. The initial pellet keeps falling apart within the eppendorf tube. We shall go back to drawing large colonies from plates. 


Methods


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) Inoculate center of plate (directly onto the plastic)

10) Dispense 100-150 microliters of chemoattractant media into one of the insertion sites

a. Use 1% agar media

11) Incubate Plates

12) Regularly Picture


Resazurin Plates

1) Create 50mg/ml Resazurin Stock Solution in PCR water

2) Filter at least 3ml of the Resazurin Stock Solution into a 15ml conical vial using a 0.22um filter and syringe

3) Melt down media (in this case 0.75% SMA with 1% Agar)

4) Let media cool to at least 50C (verify with temp gun)

5) Pipette 9ul of stock solution for every ml of media (e.g. 900ul for 100ml of media) to create a final concentration of 0.75mg/ml

6) Stir Well

7) Pour Media into Plates (Molds optional)


New Twitch Inoculate Considerations - CANCEL THIS METHOD

1) Inoculate Broth with Desired Culture

2) For each inoculate, reach an OD of 1.0

3) Place 1ml in an eppendorf tube

4) Centrifuge for 60sec 

5) Remove supernatant

6) Place Pellet at the Center of the Twitch Plate


Twitch Plate Considerations

1) Create stock of attractants at 1mM, 2mM and 10mM

2) Create three holes away from the inoculation site in twitch plate

3) Inoculate from nearest to farthest 1mM, 2mM and 10mM respectively

4) Create a concentration gradient for the cells to migrate towards


Results

        The inoculates from the eppendorf tubes kept falling apart. We ended up leaving 5ul of broth behind and pipetting those 5ul of broth into the center of the plates instead of attempting to grab a dry pellet from the bottom of those eppendorf tubes. 


Discussion

       For now, it is more important to get directed twitch than it is to standardize the initial inoculate size. Thus, we are reverting back to the old methods of drawing colonies from plates and inoculating into the center of our SMA plates.

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