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Abstract Study question Which temperature probe (thermocouple Type T or Pt100) is the most accurate and robust for measuring medium temperatures in dishes on a heated surface? Summary answer A Pt100 probe attached to the bottom of the dish provides a more accurate and repeatable measurement than the commonly used thermocouple Type T probe. What is known already The success of an IVF cycle is dependent on several variables. Ensuring the gametes and embryos are exposed to appropriate temperatures is a critical variable during incubation and manipulation. There are different temperature measurement probes available for this purpose. Their specific properties could make them more, or less suitable for the various temperature measurements required in the laboratory. Study design, size, duration The aim of the study was to determine the best method (thermocouple Type T probe placed in the medium or a Pt100 sensor attached to the bottom of a dish) to measure temperature in medium held in two types of dish placed on a heated surface. Measurements were made in triplicate and standard deviation determined. Participants/materials, setting, methods The temperature of the medium in two types of dish (5 well and 40mm direct surface contact dishes, Vitrolife) were measured with two types of probes. The commonly used thermocouple Type T, which is composed of two wires, a copper and copper nickel alloy, that are welded together, and a resistance sensor composed of a small platinum plate attached to the bottom of the dish (Pt100). Main results and the role of chance With a 100 μl droplet under 5.0 ml of oil in a 40 mm dish (with the lid on) the tip of the Type T probe measured a temperature 3.2 ± 0.26 °C lower than the Pt100 probe. The Type T probe also measured a lower temperature in the 5 well dish. With a well containing 500 μl (lid on) it measured a temperature 2.1 ± 0.15 °C lower than the Pt100 probe. When the entire length of the Type T probe was placed inside of an incubator set at 37.0 °C the measured temperature was 0.1 °C lower than the Pt100 probe. This suggests the length of the Type T probe within the environment to be measured is critical, i.e., the lower temperatures obtained with the Type T probe when measuring medium temperature on heated surfaces depends on the method used rather than the probe itself. The reason for the lower temperature is correlated to the high temperature conductivity of the copper in the Type T probe. This also makes the measurement less repeatable as different factors (e.g., length of probe submerged, ambient temperature, air flow, etc.) will affect the reading. The Pt100 is not influenced by such factors. Limitations, reasons for caution The dishes used in this investigation create a uniform direct contact with the heated surface. This means that the heated surface will have a temperature close to that measured by the Pt100. This will not be the case with dishes with an air gap design. Wider implications of the findings This investigation highlights the importance of using the correct type of thermometer probe for the purpose. It is likely that when using a thermocouple Type T probe, temperature in the medium can be 2-3 °C higher than measured, which could impact gametes/embryos and lead to deleterious results. Trial registration number Not applicable