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In many life science applications, such as growing and storing cell cultures, the carbon dioxide (CO2) incubators that house these cell cultures must maintain an ideal environment for cell growth by providing CO2 control in a humidified atmosphere with a consistent temperature. The incubators must also be adequately sealed to isolate cell cultures from external conditions and to prevent outside contaminants from affecting the sampling. This requires that the CO2 concentration, relative humidity, and temperature be measured and controlled with extreme accuracy and reliability. Choosing high-quality measurement technologies and precisely measuring CO2 levels is critical for successful incubator operation, but most importantly, for optimal cell growth.
As an attendee, you will learn more about:
- Incubator measurement methods and the importance of choosing high-quality sensors
- The challenges of achieving accurate measurements and how to overcome them
- The technology behind high-end sensors and the key benefits
- How to install measurement technology accurately and key considerations when installing
- The importance of spot-checking and field calibrations to ensure the most accurate readings
Speaker
Jacalyn Whitney
Application Sales Engineer
Vaisala
Hello everyone and welcome to lab managers ask the expert webinar series. My name is Marybeth Donna and I'll be moderating this discussion how to ensure viable outcomes with accurate co2 RH T incubator measurements. We like our webinars to be very interactive, so we encourage you to submit your questions to us at any point during today's webinar. Our speaker will address these questions during the question and answer session following her presentation. To ask a question or leave a comment, simply type your query into the q&a box located on the right hand side of your screen.
We will try to address as many questions as possible during our time together. But if we happen to run out of time, I will forward any unanswered questions to our speaker and she can respond to you directly if possible. I would like to remind you that this webinar recording will be available on demand shortly following this presentation. So please watch for an email from lab manager and how to access this free video once it's available.
Please also see the handouts tab on the right hand side of your screen for more information about this presentation. I would also like to extend a special thank you to our sponsor Vizsla who support allows lab manager to offer these webinars free of charge to our readers. So with that, I'd like to introduce our presenter for this webinar. Jacqueline Whitney is an application Sales Engineer advice Allah with four years of industry experience. She has been with Vizsla since April 2022, and worked for vacuum barrier Corporation for over three years prior to joining Vizsla.
She holds a bachelor's degree in mechanical engineering with a concentration in manufacturing and industrial design with a minor in mathematics from Merrimack college and Massachusetts. Thanks for joining us today Jackie. I would also like to introduce Carol Varadero senior marketing manager who will give a brief intro to Vizsla before Jackie begins her presentation. Carol, thanks for joining us today.
Thank you Marybeth for the introductions. We're happy to be here to present on this important topic. For those of you who are unfamiliar with Vizsla, I'll give you a quick snapshot of who we are and what we do. Vizsla. We started over 80 years ago as a global leader in the development of weather and environmental technologies, and is headquartered in Finland.
For the past 40 years, we've been leading the way in industrial measurements, bringing that expertise to a wide variety of controlled environments, including life sciences, we offer solutions equipped with powerful sensors, including human cap and cargo cap to measure parameters such as temperature, humidity, and dew point, as well as gas concentrations of co2 and hydrogen peroxide. companies and institutions around the world depend on instruments to aid in their processes, and protect the quality of their products and environments.
Our instruments safeguard and control critical processes such as in pharmaceutical, pharmaceutical manufacturing, in biotechnology research. We even have sensors on two planets is the Mars Curiosity and perseverance rovers are equipped with Vizsla, humidity, and pressure sensors to measure atmospheric conditions. Our products and services provide our customers with the means to influence and better understand their environments. I'll now turn the presentation over to Jackie, Jackie Whitney.
Thank you, Carol. Hello, everyone. My name is Jackie Whitney, thank you for tuning in to this webinar on how to ensure viable outcomes with accurate incubator measurements. Today we will be talking about what is often measured in incubators, the different measurement methods and the importance of using high quality sensors and the technology behind them. I'm assuming most of you know what a co2 incubator is.
But for those of you who need a brief refresher, co2 incubators are special climate chambers used by life science laboratories to grow or store biological samples, such as cell cultures. The environments within these chambers are typically warm and humid to keep the sensitive samples fresh and alive. Next, we will talk about all the parameters that are monitored and maintained within the sealed chambers. The conditions inside incubators must be controlled very carefully. Temperature control is vital to ensure even heating of an incubator, typically provided by a heater and a fast rotating fan that continuously mixes the air inside the cabinet.
This also helps to generate an even moisture and co2 concentration in the air volume. The most demanding element of parameter control is when pure carbon dioxide is fed from a bottle and mixed inside the incubator with the cabinet air to achieve the required concentration. In addition to temperature, relative humidity and co2. oxygen levels are also monitored within certain incubators, such as dry gas incubators. The standard incubator must maintain an average temperature of 37 degrees Celsius or or 98.6 degrees Fahrenheit, a high humidity of at least 90% and a co2 concentration of 5%.
When manufacturing and incubator, they're typically calibrated while considering the atmospheric pressure at sea level. So if you're performing your tests at a much higher altitude, like and Colorado, then it would be important to get your unit recalibrated at that elevation. Now that we've talked about what is measured in incubators next we will talk about how these parameters are measured. There are two main measurement methods, continuously monitoring measurements and calibration measurements. To ensure that the samples are in the appropriate environment throughout their cycle.
Sensors are installed inside the incubator to continuously monitor the conditions. These sensors can be installed by the incubator manufacturer, or the end user and can send the outputs to a monitoring system or building management system. Eventually, the sensors within the incubator will start to drift due to continuously being subjected to the challenging and harsh conditions. That is why it is important to take calibration measurements to check the operation of the sensors and determine whether they need to be calibrated or replaced. This can be done in the field with a handheld meter in reference points that meet the requirements of the application.
As important as it is to frequently check the operation of your sensors. It is also important to choose the most reliable and high quality sensors. Think about how much time money and resources that have gone into the life science industry. It would be a shame to let even a fraction of that go to waste due to a broken sensor that failed to properly monitor and incubator full of cell samples. These samples are extremely valuable and sensitive to environmental change. So having them be monitored by stable sensors can help reduce that risk, if maintained properly. Next, we will be talking about the challenges of achieving accurate measurements.
As we mentioned earlier, most incubators operate in a hot and extremely humid environment. What happens when you place a cold object in a drastically warmer environment or suddenly increase the temperature, the air around the object condenses and water molecules start to form, which in this case can disrupt the readings of some sensors. Depending on the technology use there are also other parameters that affect the carbon dioxide measurement. In tread measurements is based on a calculation of actual carbon dioxide molecules when the gas density has to do with the measurement.
Therefore pressure and temperature are parameters that affect measurement accuracy. Based on the ideal gas law, the sample gas expands at a higher temperature and therefore shows a lower co2 concentration than the same sample at a lower temperature. In addition, the same co2 concentration at lower pressure shows the two low readings, which is relevant for incubators operated in high altitude locations. Humidity and oxygen have a minor effect on measurement accuracy. But usually this is not significant because they out balance each other. Between operation the inside of the incubator is cleaned, meaning that the sensors inside are also exposed to these cleaning agents, and over time the cycle can wear down the exposed sensor.
The incubator environment is optimal not only for the growth of cell cultures, but also for harmful microbes such as bacteria, viruses and fungi, which can contaminate the samples if the incubator is not cleaned properly or consistently. lab personnel will either clean the interior manually with a cleaning agent, or use more drastic methods such as heat sterilization. vaporized hydrogen peroxide or UV light. It is important to consider your cleaning approach when choosing your sensors and installation methods.
Your sensor manufacturer should be able to tell you which methods are approved so that the sensors are not damaged or destroyed. Now we're going to take a deeper dive into the technology behind these high end centers and their benefits. Originally, thermal conductivity sensors were used in work by measuring the electrical resistance through the air. The sensors are typically comprised of two cells, each containing a thermal resistor or thermistor. One cell is exposed to the air of the chamber and the other cell contains a reference atmosphere that is sealed off at a controlled temperature.
The resistance of the thermistor will change as the temperature humidity and gas composition change. When the temperature and humidity is the same for both cells, the difference in resistance we'll measure the difference in the gas composition, which in this case reflects the level of co2 in the chamber. If a difference is detected, then the system is prompted to add more co2 to With the atmosphere, TC sensors are less expensive and have a less effective accuracy than infrared co2 sensors.
Because the resistance differential can be affected by other factors and just co2 levels, the temperature and humidity in the team chamber must remain constant for the system to work properly. Every time the chamber door opens, you must wait to take readings until the atmosphere stabilizes, which could take roughly 30 minutes or so. Infrared sensors are the more expensive but reliable and stable option. They rely on the fact that each gas absorbs a distinct wavelength of light. co2 absorbs a 4.3 micro meter wavelength which is within the infrared portion of the electromagnetic spectrum.
I will talk more about how these sensors work over the next couple of slides. There are a few different ways to build these infrared co2 sensors. But all it really comes down to a light source, filter and detector all placed within an optical sensor structure called the cuvette. an IR emitter directs infrared light through a sample of the growth chambers atmosphere, then through filter which isolates the proper wavelength and finally into a sensor. Periodically calibrated circuitry measures the amount of 4.3 micro meter light that strikes the sensor and calculates the difference between it and what was emitted by the source.
The more co2 The less light passes, the difference allows the circuitry to calculate the percentage of co2. Because the light absorption does not depend on temperature humidity, the center is accurate anytime, including shortly after opening and closing the door of the growth chamber. Just like any sensor, the readings can drift over time. With the sensors it can happen if the intensity of the light source changes, contaminants get in the way of the optical cuvette or if the sensor undergoes any other mechanical change.
This has influenced manufacturers to take different approaches with the light sources, filters and detectors. The generic sensor structure of one light source one filter and one detector is very sensitive to drift. But one of the main reasons ways to compensate for drift is by adding components. This can be done by either having an additional light source or reference as shown in the top image or an additional filter to reference as shown in the middle image. Both options are better than generic than the generic structure. But the added reference components can also deteriorate over time and lose their effectiveness. The third image reflects how we build our co2 carbo cap sensors, which is with one IR source, one tunable filter in one detector that can identify both the co2 absorption and reference wavelength, which gives us true internal referencing and long term stability.
The tunable bandpass filter also known as the Fabry Pirot and Ferro meter filter, is what allows us to take in both the co2 absorption wavelength and reference wavelength and continuously alternate between measuring the to the co2 concentration is then calculated from the ratio of the two signals. Because the detector is measuring both wavelengths with the FBI filter, it makes the reading insensitive to drift Okay, time for a quick pop quiz. This should be an easy one.
What environment most accurately emulates that of an incubator environment. A a dark basement, be the human body, see outer space, or D a refrigerator? So I'll give you guys a few seconds to answer this one. And it looks like you're all getting a similar answer. And the answer is B the human body. Now we're going to switch gears and talk about humidity sensors. As mentioned before incubators must maintain a high humidity while running because it protects the specimens from drying out.
In an ideal case, the humidity inside the cabinet would be close to 100%. To keep the samples fresh and alive, though relative humidity is often closer to 90% because of the temperature gradients and the limited accuracy of moisture control. The simplest incubators contain a water pan on the cabinet floor to allow water to evaporate in the air. If the water is slightly colder than the rest of the incubator Condensation Condensation can be avoided. But it is challenging to maintain the humidity at an optimal level.
For example, opening the door will cause the humidity level to fall and it can take a long time to recover. Still water also presents a potential contamination risk if impurities appear in the water pool. Another solution is to use a humidity controlled at top atomizer to generate an exact amount of water particles, which requires an extremely accurate humidity measurement.
This leaves users with two options when selecting the correct humidity center for their application. They can go with inexpensive and disposable sensors that are not as accurate and quickly become unstable when exposed to these challenging environments. Or go the more expensive route and choose a sensor that can withstand the very humid environments and potentially harmful cleaning agents. Some sensors can warm up and purge residue that builds up on its surface over time. In addition to considering what sensor you need, it is also important to consider how you will install your sensor. As we know the incubators must remain closed and sealed throughout operation to maintain the controlled environment.
We must ask ourselves if we want the sensor to be permanently installed removable or used to take samples outside of the running incubator by incorporating additional accessories. If installed inside the incubator, the sensor must be positioned away from any gas and lit hotspot or cold spot that could tamper with the readings. It should be easily accessible and cannot be blocked by any other object within the chamber. It is also essential to seal in sense seal the sensor thermally from the external environment using a feedthrough seal.
If it is not properly sealed, it will form a cold spot causing humidity condensation to form inside the sensor and generate unwanted water. In general, it is important to isolate the cabinet from the external environment to avoid cold spots and free water. For sealing also offers a potential surface for contamination growth and hamper the cleaning. Depending on the type of incubator you have, this can limit your installation options. heat sterilized incubators are extremely hygienic and easy to clean because the sterilization is activated at the touch of a button when you choose when choosing a sensor, if you plan on keeping it inside the chamber while cleaning, you should make sure the sensor can withstand high temperatures and have a heating feature that prevents condensation.
The photo below shows one rare but possible installation option, which is permanently installing the sensor through the side of the incubator. This would require very precise measurements and a careful install. But once it is there, it saves plenty of space that the full probe would take up. Next we will talk about the most common incubator the all cell culture incubator compared to the heat sterilized incubators this one does not require as many central sensor features, but that does not take away from the importance of using installing the correct sensors.
Depending on the available space, you could choose a compact standalone probe with a long term stable robust sensor that is easy to maintain and integrate. accessories such as a flat cable allow you to close the door of the incubator with the probe still inside. Installation mounts and flanges are also good accessories to help secure your probe in place.
As mentioned in the previous slide, here's a photo example showing the flat cable. Another accessory that is vital for the sampling method is the pump pumping an air sample from the cabinet into the sensor with the help of a tubing of tubing and a pump. The sampling method may suffer from water condensing into the sample tubing, which may itself also cause an increased risk of contamination as tubing is hard to keep clean.
Whereas in the in situ method, the sensor can be removed from the incubator for cleaning. The sampling pump may generate sharp pressure shocks for the co2 sensor and the measurement output may fluctuate and be interrupted by pressure bursts. When the sensor is located inside the cabinet, it is in an in situ measurement, which is often the preferred option because it requires fewer components and it has a faster response time.
The sensor though must always withstand high humidity and temperature. Another thing to consider with this method is that the co2 concentration reading will be slightly higher than expected compared to the wet sample when using the pump with the drying Nafion membrane to bang as shown in the bottom left image. This phenomenon is due to a thing called dilution co2 Your density is diluted in the incubator by the volume that the water vapor occupies.
If water vapor is removed from the sample, the fractions occupied by other gases, including co2 will increase accordingly. The table below shows the dilution coefficients for the gas concentration when drying a gas sample. Dew point of the gas sample in the incubator is chosen on the horizontal axis and the dew point of the gas sample at the measurement point is chosen on the vertical axis.
Dew point of the gas samples at the measurement point can be determined with a humidity probe. To ensure your reading stay accurate, it is extremely important to check your instrument visually at regular intervals and determine whether the probe needs cleaning or filter replacement. The next step is to check the co2 calibration with a reference at regular intervals. This can be done by using a reference bottle of gas meter or by sending the probe back to the factory for a thorough health check and calibration.
Many labs have a handheld meter available to do these checks. This photo shows a common setup that we recommend to those in the life science industry for their spot and calibration checks. Here we have the handheld meter connected to a co2 and relative humidity and temperature probe with the flat cable and probe holder. One of our customers Boston IVF uses our GM 70 handheld carbon dioxide meter to verify the conditions of their IVs incubators daily.
This meter was shown in the previous slide and as well on the right side of this slide. Since 1986 reproductive treat reproductive treatment through Boston IVF has resulted in 90,000 births. Boston IVF embryo incubators are one of the most important pieces of equipment at their facilities. They specifically monitor temperature, humidity and pH to ensure an ideal environment for the embryos. The pH levels during incubation are an important measurement obtained by monitoring the concentration of carbon dioxide in the chambers because pH levels are dependent on the co2 levels.
However, as with any parameter and indirect measurement needs, a sensor needs to be highly accurate to de emphasize uncertainties in the measured value. Daily checking of the chamber with Vizslas handheld meters ensures that any drift in the accuracy of incubators built in sensors is detected immediately. Most incubator sensors are calibrated to a single point. So a more accurate measurement is required to make any needed adjustments of co2 levels. This concludes our presentation. Thank you for joining. And now I will open the floor to some questions.
All right, great. Thanks, Jackie, for a wonderful presentation. So at this point, we are going to move on to the question and answer session of this presentation. Again, for those of you who may have joined us late, you can send in your questions by typing them into the q&a box located on the right hand side of your screen.
Even if you don't have a question, we invite you to leave a comment let us know how you enjoyed this presentation. We will also forward any comments to the Vizsla team. If we do not have time to get to your question. Or if you would like them to get back to you. We will have a record of your name and email address if you leave a comment, and they've also provided their contact information on the screen in front of you. And there are some handouts available for you in the handout section. So Jackie, I'm going to turn your mic back on. Thanks again for a great presentation. Let's go to the first question here from the audience. This one says when spot checking using a handheld How long should I let the reading stabilize?
So that's a good question. A good rule of thumb is to is to wait about 30 minutes for to stabilize. But you can use the graphical display of the indicator to see when the curve has flattened.
Okay, great. Thanks. We have another question here. This one says Do you have any recommendations for cleaners for the sensors?
We typically recommend alcohol based cleaning agents. Some examples are ethanol and 70% isopropyl pull alcohol as well as acetone and acetic acid can commonly be used for co2 sensors as well. Okay, great, thanks.
Here's another one that came in what is the calibration interval of Vizslas sensors?
So it depends on how often and how frequently you're using the sensor and what kind of environment you are exposing it to. But we generally recommend every 12 months if you maintain it properly. This can be done by on your own by using a reference source and a handheld meter. Or you can send the instrument to our lab to be calibrated.
Great, thank you. This next one says is it possible to spot check two incubators at once?
Yes, so our Mi 70 meter has two pro ports. So once they are connected and the indicator is turned on, you can go into the display settings to adjust which measurements are shown on the main screen. If you're using both probes to measure the same condition, like you would in incubator applications, you must first check environment settings to ensure they are the same for each port. Then when measuring humidity and co2 simultaneously, you can use the Hmp 70s probe temperature data to compensate the co2 measurement. The display will then identify which reading is coming from which port by using a little one into symbol when in use.
Okay, wonderful. Thank you. Here's another question. This one says how do you prevent condensation from forming in the tubing when pumping a sample.
So, I pre fill previously spoke briefly of this. So by having part of the sample tubing be made of a material called Nafion, which is highly selective in the removal of water. They beat it basically works by the water moves through the membrane wall and evaporates into the surrounding air in a process called pre PIR evaporation. Nafion removes water by absorbing absorption which occurs as a first order kinetic reaction. In drying applications, the moisture exchanger transfers water vapor from a wet gas stream into the surrounding atmosphere. Drying is complete when the sample humidity level is equal to the ambient humidity level. Since drying proceeds as a first order kinetic reaction, this level can be reached extremely quickly, usually within 100 to 200 milliseconds.
Great, thanks. I think we have time for one more question here. This one says if using an Rh T and a co2 probe, is there a minimum distance they should be spaced?
Yes, so the co2 probe has some slight warming, so it can affect the relative humidity and temperature reading. So you shouldn't velcro or zip tie these two together when installing it into your incubator. But we do offer an accessory for this purpose, which was shown on the typical setup slide is keeping the probes at a short distance apart at all times.
Okay, wonderful. Thanks so much.
Thank you.
So that does bring us to the end of this webinar. And just a reminder that this webinar will be available on demand shortly following this presentation. Please watch your email for a message from land manager on how to access this free video once it's available. So on behalf of lab manager, I'd like to thank Jackie Whitney for all the hard work she put into this presentation. And I would like to thank all of you for taking time out of your busy schedules to join us today. Once again, thank you to our sponsor Vizsla whose support allows us to offer these webinars free of charge for our readers. For more information on all of our upcoming or on demand webinars or learn more about the latest tools and technologies for the laboratory, please visit our website at lab manager.com. We hope you can join us again thank you and have a great day