Laboratory Procedure and Pipetting Instructions

Participants should pick up the tubes, strap them, and place them in the provided black rack. Each participant will have their own black rack.
A total of four tubes should be placed in the rack.

Each tube should be labeled on the frosted section using initials for easy identification.
It is important to ensure that tubes are capped properly before proceeding to the next steps.

Goggles are required when starting the process involving projectiles or pipette headers.
Participants must wear their goggles before moving on to the next steps.

The pipette to be used for the next step is the p 1,000, which should read 090 on the display, indicating it can handle 900 microliters (0.9 mL).

Familiarize yourself with how to use the pipette:
- Soft Stop: Press down until you feel resistance (first stop).
- Hard Stop: Push all the way down to release liquid.
Steps to pipette:
1. Dip the tip into the water (do not push it all the way down).
2. Release the plunger to fill the pipette tip with 900 microliters of water.
3. Withdraw the pipette and dispense the water into tube number one.
Repeat the process for all four tubes, aiming for consistent volume among all tubes.
If the tip touches anything outside of the liquid, it is necessary to change the tip to prevent cross-contamination.
Ensure that all tubes have approximately the same volume of water.

After filling, verify the volumes in the tubes before proceeding:
- Ensure all tubes have the same amount of liquid (indicated by a mark on the tube indicating the desired level).
- Check for air bubbles that may be affecting the volume and adjust as necessary.

Each pair will have access to a digital scale which should be turned on for weighing.
Participants must place their weigh boat on the scale carefully, ensuring fingers do not touch the inside of the weigh boat to maintain accuracy.
- Important to press the tare button to reset the scale to zero before weighing.

Use the spatula provided to fill the weigh boat with soil:
- Aim for approximately 1.0 gram of soil.
- The precision of weighing can vary based on the type of soil.
- If too much soil is added, adjust by scraping some away without touching the weigh boat with fingers.
- Acceptable variation is when the scale fluctuates between values close to 1.0 gram.

After all soils have been measured:
- Each participant will use a vortex mixer to combine water and soil.
- Hold the vial securely, ensure the cap is tight, and vortex for 5-10 seconds to mix well.
- Expect the resultant mixture to contain microbes and be evenly blended.

Pipetting Tips: The pipetting will switch to using blue-bottomed pipette tips. Ensure unused tips and pipettes are stored properly with lids closed.
Prepare to transfer only from the second, third, and fourth tubes to new plates:
Allocate 100 microliters from each tube to the designated plates (plate 2, plate 3, and plate 4).
Use the same pipetting technique:
1. Soft stop to fill the pipette tip with liquid.
2. Inserting the tip into the new location (e.g., nutrient agar on the plates).
3. Use a cell spreader to evenly distribute the liquid on the plate surface.
Ensure to use a new tip for each transfer to prevent contamination.

It is critically important to label all plates clearly:
- Initials and the date must be included (e.g., “Initials – 10th February”).
- Clearly label positions: e.g., Plate 2, Plate 3, Plate 4 based on the tube sources they originated from.
After pipetting, ensure all waste and materials are disposed of properly and station cleanup is done after the experiments.
Finally, return the pipettes and tips to their original locations as specified.