Using the Deep 96-Well Pin Tool

The 96-well pin tool can be used to transfer long-term evolution experiments and to make dilutions when plating many samples. Although advertised to transfer 5 µl of liquid, it routinely transfers ~3.5 µl of culture in my hands. It also appears to be slightly less precise (~10% s.e.m.) than a pipetman (~5% s.e.m) based on replicate test dilutions of identical cultures.

Preparation

1. Fill two large tip boxes with 10% bleach and ddH₂O. The level of liquid in the bleach should be high enough to reach about halfway up each pin when the pin tool rests on the sides of the tip box. This is approximately 400 ml. The level of the ddH₂O should be 0.5 cm higher, about 450 ml.
2. Fill the pyrex dish with isopropanol (2-propanol). The level of the isopropanol should be as high as possible, almost to the top of the pins when resting the pin tool in the tip box. Keep the isopropanol reservoir furthest from the flame to avoid accidental fires. If it does catch fire, try putting the plastic lid down to suffocate and extinguish the flame. If that doesn't work, use an ice bucket filled with room temperature water.
3. Place a sheet of lint-free filter paper on top of two folded paper towels. Use the same sheet of lint-free paper over multiple days until it begins to disintegrate.

Each Use

1. Pickup - Mix the sample by moving the pins around the wells several times. Move slots in pin tool up and down through the meniscus of the samples to be transferred at least three times. When pulling the pins out the final time, do it slowly and deliberately.
2. Transfer - Smoothly move the liquid-filled pins to the next plate, avoiding any jarring motions that might send drops flying. Dip the pins into fresh media and move slots up and down through the meniscus at least three times. Remove the pin tool deliberately to avoid any splashing from the pins as it is removed from the fresh plate.
3. Clean - Wash once with 10% bleach, then twice with distilled water. Each time moving up and down at least three times through the meniscus and then blotting on the filter paper. Dip into isopropanol at least three times an then flame in a Bunsen burner. Rotate the tool while it burns so that all of the pins are engulfed in flames. After the flame goes out, wave the pin tool quickly through the Bunsen burner flame one additional time to catch any leftover isopropanol in the slots.

Mixing

• Before plating any competitions, it is important to thoroughly mix the 96-well plate with the pin tool. Clean the pin tool, and place it in the plate. To mix, move the pin tool in a circular motion and up and down, passing through the meniscus each time. The motion is similar to that of churning butter, and the motion should be repeated in various directions until each well is mixed.

Important Considerations

• The speed with which you remove the pin tool from the liquid when transferring determines the volume that it picks up. The quicker you remove it, the larger the drops on the end of each pin will be. Remove it as slowly as possible to get reproducible results and avoid contamination.
• On the other hand, if you want to use the pin tool to spot drops of liquid on a petri dish, it is helpful to remove the tool more quickly to form a larger drop that hangs from the pins. Note that only 1-2 µl, rather than 3.5 µl will be spotted; a considerable amount of the volume will remain in the slot of each pin.