Setting Up the ICSI Dish : Oil Overlay and Drop Management

Monash Biotech

December 17th, 2025

In the IVF laboratory, the ICSI dish is not merely a container; it is a micro-environment. For the 15 to 20 minutes an oocyte spends on the heated stage, that dish is the only barrier between a viable gamete and a hostile environment.

While the injection technique itself gets the glory, the setup of the dish is where the battle for stability is won or lost. A poorly prepared dish leads to osmolality drifts, pH spikes, and temperature fluctuations—stressors that can depolymerize the meiotic spindle before you even pick up the injection pipette.

This protocol outlines the best practices for drop management and oil overlay, ensuring your media remains stable from the first oocyte to the last.

1. The Physics of the Microdrop

The challenge in ICSI is volume. We work with micro-volumes (typically 2–5 µL drops). Without protection, a 4 µL drop of medium will undergo significant evaporation within seconds at 37°C.

• The Consequence: Evaporation removes water, increasing the concentration of salts.This hyper-osmolality draws water out of the oocyte, causing shrinkage and hardening of the zona pellucida, making injection difficult and lysis more likely.

To prevent this, the Oil Overlay is non-negotiable. It acts as both a vapor barrier and a thermal buffer, smoothing out the rapid cooling that happens when a dish is moved from the incubator to the microscope stage.

2. Step-by-Step Drop Management

Step A: Design Your Map Before opening the sterile pack, have a consistent "Dish Map." Most embryologists prefer a central row of media drops (Buffered HEPES or MOPS) for oocytes, flanked by PVP drops for sperm manipulation.

• Pro Tip: Keep PVP drops and Media drops sufficiently spaced. If they are too close, the oil overlay process can drag the viscous PVP into the media, contaminating the oocytes.

Step B: Pipetting the Drops

• Deposit the drops vertically.

• Do not "smear" the drop on the plastic. A round, high-domed drop offers better optical properties than a flattened, smeared one.

• Immediate Cover: Do not pipette 10 dishes in a row and then oil them. Pipette one dish, oil it immediately. Speed is critical to prevent initial evaporation.

Step C: The Oil Overlay Technique Pouring oil is a skill.

1. Pre-warm the oil. Cold oil on warm media causes immediate temperature shock.

2. The Gentle Pour: Do not pour directly onto the drops. Pour the oil into the edge of the dish and let it gently flood across the surface, covering the drops like a rising tide. This prevents "exploding" the drops or creating micro-bubbles that interfere with optics.

3. Volume Matters: Ensure the oil completely covers the drops with at least 2-3mm of headspace. A thin oil layer offers poor thermal protection.

3. The Hidden Variable: Optical Clarity and Flatness

Once the dish is on the inverted microscope, the plasticware becomes part of your optical train. You are looking through the eyepieces, through the objective, through the glass stage, and finally through the bottom of the plastic dish.

If your dish is low-quality, two problems arise:

1. The "Warped Floor" Effect: Cheap plastic often has microscopic unevenness. As you move the stage from the sperm catch drop to the injection drop, the focal plane shifts. You are constantly adjusting the focus knob, wasting time and motion.

2. Refractive Interference: Poor-quality polystyrene creates a "haze" or birefringence under Hoffman Modulation Contrast (HMC) or DIC optics. This makes the polar body look fuzzy and the oolemma indistinct.

The Solution: Genesis 50mm ICSI Dishes

At Monash Biotech, we engineer our Genesis 50mm Dishes specifically for high-magnification micromanipulation.

• Why 50mm ? It provides the perfect surface area—larger than a 35mm dish (allowing for more PVP drops/sperm separation) but low-profile enough for standard stage warmers.

• Optical Grade Polystyrene: We use crystal-clear, USP Class VI virgin polystyrene.The refractive index is matched to standard microscopy requirements, ensuring that when you look at the oocyte at 400x, the edges are razor-sharp.

• True-Flat Bottoms: Our molding process guarantees a planar surface. When you slide from the PVP drop to the oocyte drop, your focus remains locked.

Conclusion Don't let a generic petri dish compromise your high-precision work. Control your temperature with oil, control your osmolality with speed, and control your view with Genesis.

Disclaimer

Note to Professionals: Monash Biotech is a manufacturer of high-precision ART consumables.The protocols described here are for educational purposes. Always follow your laboratory's specific Standard Operating Procedures (SOPs) regarding media volumes and oil types. Consult your Lab Director before changing plasticware brands.