How to Get Rid of Bubbles in 96 Well Plates

Bubbles in 96-well plates can disrupt experiments, skewing results and leading to inconsistent data in biological and chemical assays. These bubbles often form during liquid handling, pipetting, or mixing reagents, creating uneven distribution and inaccurate readings. Addressing this issue is crucial for researchers and laboratory technicians who rely on precise measurements for high-throughput screening and diagnostic tests. By understanding how to get rid of bubbles in 96 well plates, labs can ensure better accuracy and repeatability in their experiments. This article explores the best practices, alternative methods, and important considerations to eliminate bubbles efficiently and effectively.

Best Ways to Get Rid of Bubbles in 96-Well Plates

1. Slow and Controlled Pipetting

One of the most effective ways to reduce bubble formation is to pipette liquids slowly and with control. Rapid pipetting can introduce air, causing bubbles to form at the bottom of wells. By angling the pipette tip against the side of the well and allowing the liquid to flow gently down, you can minimize bubble formation. For example, when adding reagents to sensitive ELISA assays, slow pipetting ensures even distribution without disrupting the assay surface. Consistent practice with controlled pipetting techniques can significantly reduce errors in liquid transfer.

2. Pre-Wetting Pipette Tips

Pre-wetting pipette tips before transferring liquids can help reduce bubbles in 96 well plates. This involves aspirating and dispensing the liquid once or twice before the actual transfer. Pre-wetting coats the inner surface of the tip, reducing the likelihood of air pockets forming during dispensing. This method is particularly useful when handling viscous liquids, such as protein solutions, that tend to introduce bubbles more easily. In PCR preparations, pre-wetting ensures accurate reagent dispensing, preventing the formation of bubbles that could impact amplification efficiency.

3. Using a Plate Spinner or Centrifuge

A plate spinner or low-speed centrifuge can remove bubbles by applying centrifugal force, pushing air bubbles to the surface or edges of the well. This method is especially beneficial after pipetting viscous liquids or when preparing 96 well plates for spectrophotometry. By briefly spinning the plate at low speeds (e.g., 500 to 1000 rpm), bubbles that may have formed during mixing are effectively expelled. For example, cell culture plates often benefit from this technique to ensure cells are evenly distributed without trapping air pockets. This simple step can dramatically improve the consistency of results.

4. Gently Tapping or Shaking the Plate

After dispensing liquids, gently tapping or shaking the 96 well plate can dislodge trapped bubbles. Light taps on the side or bottom of the plate encourage bubbles to rise to the surface and burst naturally. This technique is quick and effective, especially for quick assays or when using high-throughput processes. However, it requires careful handling to avoid spilling or cross-contamination. Tapping is often used in enzyme-linked immunosorbent assays (ELISA) to ensure uniform liquid layers across wells, improving overall assay performance.

5. Utilizing Bubble Removal Tools

Specialized bubble removal tools, such as bubble paddles or vacuum-based devices, are designed to extract bubbles from wells. These tools are useful when working with sensitive assays where bubbles could interfere with light transmission or fluorescence readings. For instance, microplate readers measuring absorbance require clear wells for accurate results. Bubble removal paddles allow technicians to glide over the liquid surface, eliminating surface bubbles without disturbing the sample. Investing in these tools can lead to better reproducibility in high-throughput screening experiments.

6. Applying Heat or Vibration

Mild heat or vibration can reduce bubble formation by decreasing the surface tension of the liquid. For example, placing the plate on a vibrating platform can gently agitate the liquid, allowing trapped air to escape. In cell culture applications, incubators with vibration settings can aid in bubble elimination without harming cells. This technique is also used for PCR master mixes, ensuring bubbles do not interfere with the sealing process or subsequent amplification steps. Care must be taken to avoid overheating, which could compromise sensitive samples.

7. Adjusting Liquid Volumes

Overfilling or underfilling wells can lead to uneven liquid distribution and increase bubble formation. Ensuring that liquids are dispensed at the correct volume, typically between 50 to 300 µL depending on the well size, reduces the chance of introducing bubbles. Automated liquid handling systems can be programmed to dispense exact volumes, minimizing manual errors. For example, in drug screening assays, precise volumes are critical to ensure consistent drug concentrations across wells. Maintaining accurate volumes contributes to more reliable and reproducible data.

8. Using Surfactants or Additives

Adding small amounts of surfactants, such as Tween-20, to reagents can lower surface tension and reduce bubble formation. This method is commonly used in immunoassays and cell culture media. Surfactants help liquids spread evenly across the well surface, preventing bubble entrapment during dispensing. For ELISA procedures, a 0.05% Tween-20 solution is often used to coat wells and minimize liquid beading and bubble formation. This simple chemical adjustment can enhance assay performance and reduce variability between wells.

Other Ways to Get Rid of Bubbles in 96 Well Plates

1. Manual Removal with Pipette Tip – Gently use the pipette tip to nudge bubbles to the surface, allowing them to burst.

2. Vacuum Desiccation – Place the plate in a vacuum chamber to draw out trapped air bubbles from the wells.

3. Blotting the Surface – Lightly press an absorbent material over the surface to remove bubbles without disturbing the liquid.

4. Blow Gently with Air – Use a fine air stream to break surface bubbles, ensuring not to introduce new air pockets.

5. Hydrophobic Coating – Pre-treat the plate with hydrophobic solutions to prevent bubbles from adhering to the well surface.

Things to Consider When Removing Bubbles

1. Sample Sensitivity:
Certain biological samples, such as live cells or delicate proteins, can be damaged by aggressive bubble removal methods. It is crucial to choose gentle techniques, like tapping or slow centrifugation, to avoid compromising sample integrity. Heat or harsh vibration may lead to cell death or protein denaturation, affecting assay outcomes. Always consider the nature of the sample before applying bubble removal techniques.

2. Assay Type:
The type of assay being performed determines the best bubble removal method. Fluorescence-based assays, for example, are highly sensitive to bubbles that can interfere with light transmission. In contrast, simple colorimetric assays may tolerate minor bubbles without significant impact. Knowing the sensitivity of the assay allows for the selection of the most appropriate bubble removal strategy.

3. Well Plate Material:
Different well plate materials, such as polystyrene or polypropylene, exhibit varying levels of hydrophobicity. Hydrophobic surfaces are more prone to bubble formation, requiring additional steps to ensure smooth liquid dispersion. Pre-treating the plates with surfactants or plasma treatments can help reduce this effect. Understanding the properties of the well plate material can prevent unnecessary bubble formation.

4. Volume and Pipette Calibration:
Incorrect pipette calibration can lead to inconsistent volumes and introduce bubbles. Regular calibration ensures that dispensed volumes are accurate and bubble formation is minimized. This is particularly important for high-throughput assays where large volumes of plates are processed daily. Consistent maintenance of pipetting equipment is essential for reliable results.

5. Environmental Conditions:
Temperature and humidity can influence bubble formation during liquid handling. Warmer temperatures may reduce surface tension, while cooler environments may encourage air entrapment. Laboratories should maintain stable environmental conditions during pipetting and liquid handling processes. Monitoring these factors helps ensure consistent liquid behavior and minimizes bubbles.

In Summary

Eliminating bubbles in 96 well plates is essential for producing accurate and reliable experimental results. By incorporating controlled pipetting, plate spinners, and bubble removal tools, researchers can significantly reduce errors and enhance data quality. Implementing the strategies outlined in this article ensures that assays, from ELISA to PCR, remain free of bubbles, leading to more consistent and reproducible findings.