Table of Contents

Mark's Noob Proof Bleaching Worms for Eggs Protocol™

[JLO- 9/2/2026]

Overview

Materials

Bleaching Protocol

Making the Bleaching solution

270mL Bleach Solution Recipe(for egg bleaching with 42 HG Chunk plates): 

      90mL NaClO (bleach)
      15mL 5N KOH
      165mL ddH20

20mL Bleach Solution Recipe(for egg bleaching with two HG Chunk plates): 

      6.6mL NaClO (bleach)
      1.12mL 5N KOH
      12.2mL ddH20

1) Clean benchtop where you are going to make the worm egg bleaching solution

2) Retrieve materials (ALL MUST BE STERILE IE. MARKED WITH BLACKENED AUTOCLAVE TAPE): 

      Bottle of NaClO from 4C Fridge
      5N KOH from Shelf Near G20 Centrifuge
      Baxter Water from Shelf Near G20 Centrifuge
      [1] 50mL Conical Tube

3) Pipete reagents using sterile technique and while using gloves and eye protection

Protocol for two High Growth (HG) TJ1060 Chunk plates

1) Clean benchtop near centrifuge and vacuum pump with 70% Ethanol

2) Retrieve Materials

3) Using a 5mL pipette add 2.5-4.0mL of M9 to the one of the two HG chunk plates. Lightly swirl the plate to lift the worms off of the plate. Using the same pipette suck up the M9 and place it in a 15mL polypropylene tube. Label this polypropylene tube “Eggs-(dateofbleaching)”

4) Replace the 5mL pipette with a new sterile 5mL pipette. Then add 2.5mL-4.0mL of M9 to the second HG chunk plates. Like in step 3, lightly swirl the plate to lift the worms off of the plate and using the same pipette suck up the M9 and pipette it into the 15mL polypropylene tube labeled “Eggs-(dateofbleaching)” from step 3. Make sure the 15ml “eggs” tube does NOT EXCEED 8mL

5) Spin at 1000RPM for 30 seconds. The centrifuge in G20 is set to centrifuge for 60 seconds and does not automatically stop after 30 seconds. So you need to manually stop the centrifuge from running by pressing STOP after 30 seconds. After this aspirate off the M9 using aspirator (vacuum pump).

6) Add 7ml bleach per tube; vortex tube on max settings for at least 30 seconds this is critical, make sure you set a stop watch or count how long you vortex; Centrifuge at 1500RPM for 30 seconds; aspirate off bleach.

7) Add 7ml bleach again; vortex tube on max settings for at least 30 seconds this is critical, make sure you set a stop watch or count how long you vortex; Centrifuge at 1500RPM for 30 seconds; aspirate off bleach.

8) Fill to line (15ml) with M9; make sure pellet is resuspended by tapping bottom of tube on bench / vortexing gently; Centrifuge at 1500RPM for 30 seconds; aspirate off M9. Repeat two more times.

9) Using a 5mL pipette tip, pipette 2mL of M9 into the 15ml polypropylene “Eggs-(dateofbleaching)” tube.

10) Using a P1000, pipette 1000uL of the resuspended egg-M9 solution into a new 15ml polypropylene tube labeled “M9 RESUS eggs”. Repeat this step once more but label this tube “M9 RESUS eggs tube #2” so that you have two tubes of rinsed eggs resuspended in 1mL of M9

11) Go to an open light microscope, ethanol the benchtop and the microscope staging area where you are going to put look at the eggs.

IMPORANT NOTICES: Each and every time you are going to pipette the eggs suspended in M9, you must vortex the tube for at least half a second or until the egg pellet disappears or else you will not see many eggs in your droplets as most of them will precipitate toward the bottom of the tube. Also you must be quick to pipette after vortexing or eggs will precipitate to bottom of tube! Also, pipetting from the top of the tube vs pipetting from the bottom of the tube, will pipette varying amounts of eggs, so try to pipette from the same spot/height within the tube to avoid variability in pipetting.

12) Grab a P10 pipetter and aglass slide; pipette 10uL of egg-M9 solution onto the glass slide three times to have three droplets each 10uL

13) Count how many eggs are in each droplet. Then add the total number of eggs together, then divide by three to get the average number of eggs per droplet. Once you have the average amount of eggs per droplet, find the average amount of eggs per 1uL.

CRITICAL NOTE: We want all of our plates to have about 15 eggs per plate.

For example: Droplet 1 = 100 eggs per 10 uL| Droplet 2 = 100 eggs per 10 uL| Droplet 3= 100 eggs per 10 uL| Total= 300 eggs per 30uL

Average= 100eggs/10uL | Eggs per uL average = 1 egg/1uL | Goal= 1.5 eggs/1uL

Now use this Dilution equation, M₁V₁ = M₂V₂, to dilute your egg-M9 solutions to 1.5eggs/1uL

M₁ is your starting egg per uL concentration (1egg/uL)

M₂ is your goal egg per uL concentration (1.5eggs/uL)

V₁ is your starting volume (1mL)

V₂ is your ending volume and is what you are trying to solve for

CRITICAL: We want all of our plates to have about 15 eggs per plate. So if your M₂ = 1.2eggs/uL you must pipette 12.5uL to get up to 15 eggs

14) Rearrange the Dilution equation so that you are solving for V₂; Once you have your final volume, dilute with enough M9 to reach the desired goal volume

*in the case of the example above solving for the final volume, V₂= 6mL, so you would add 5mL of M9 to each “M9 RESUS egg” tubes*

Plating Bleached Eggs

1) ethanol an empty benchtop and use flint to turn on a Bunsen burner

2) grab your seeded small lifespan plates and set them up in stacks of 5.

3) vortex the “M9 Resus egg” tube; starting from the bottom of a stack of 5, remove the top part of the plate lid and pipette 10uL of M9-egg solution onto the plate, doing your best to go slow and pipette near the Bunsen burner to avoid contamination

4) once all plates have eggs, record the time and date you finished egg plating in your lab notebook, and rubber band the plates in stacks of 5 so they cannot spill and fall apart

5) store the plates with eggs in the 25C incubator and record the date and time that you put them in the 25C incubator

Notes