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Cell culture CO2 incubators: Overview and comparison

Which functions and specifications should an incubator have ?

Of course an incubator should regulate temperature, humidity and CO2 content!
But is that really all it should do ? No!

There are many features one should take into account especially when purchasing a new incubator. New and well-designed incubators can make cell culture easier, safer and more reliable because they have features not present or not of high quality in all incubators on the market. Our overview and comparison of incubator manufacturers will help you to ask the right questions and to judge which features are important for you. The most important criteria are:

Cell culture incubator

How important these different features are for you mainly depends on whether you word in a research lab, an industrial or pharmaceutical testing or production lab or a lab where an ATMP (advanced therapy medicinal product) or TEMP (tissue engineered medicinal product) is produced. A ranking of decision criteria, that we find are a good basis for the comparison of manufacturers is listed at the very end of this page.

 

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Temperature variation and recovery after door opening

Principles of incubator heating

For cell culture incubators, two main principles are used to heat the chamber and to keep the temperature more or less constant:

Water jacket incubators

Advantages: better temperature stability, lower variation after door openings, longer temperature stability upon power failure

Disadvantages: usually, high temperature sterilization is impossible, the incubator is heavier and harder to move, slow temperature recovery after very long door openings (> 15 min), Maintenance includes water exchange or refill

Air jacket incubators

Advantages: high temperature sterilization is possible, the incubator is lighter and easy to move, faster temperature recovery after very long door openings (>15min), maintenance without water exchange

Disadvantages: lower temperature stability, fast temperature drop upon door openings, short temperature stability upon power failure

Incubator temperature measurement

Recovery after door opening - measurement methods

For the comparison of technical data from different cell culture incubator manufacturers, one can usually look up values collected during the mandatory normative type examination test (e.g. product characterization test, DIN Baumuster-Prüfung). Data is either given in the instruction manual, technical specification sheet or has to be inquired from the sales department.

German manufacturers and so some others validate the recovery of temperature after door opening (specification) of their incubators according to the DIN standard for incubators and list the recovery time in min. Here, it is important to know that this DIN standard (DIN 12880) was revised in 2007 and changes were made to the parameters and the analysis.

The new version is DIN 12880:2007-05. Important for the comparison is that due to the changes in the measurement method, values acquired according to the new method give longer recovery times. Additionally, in larger incubators like those for cell culture more readings are required: 27 instead of 9 readings. The values are therefore, more precise.

The different measurement set ups are depicted in the two schemes on the right. Top (old) and bottom (new).

According to the old version, the temperature recovery time after a 30s door opening was reached as soon as the temperature once rose above the lower temperature fluctuation limit. As shown this results in very short recovery times. 

According to the new standard, the temperature recovery after a 15s door opening is reached only when the temperature fluctuation does not any more leave the temperature range between the lower and upper fluctuation limit. So on the one hand, the door opening time was shortened and on the other hand, the recovery is defined much more strictly. As a consequence, values acquired with these two methods can not be compared.

In addition to this, one should check whether the incubator of interest has a separate door heating system (all six walls are heated). This reduces temperature gradients and condensation at the door. In comparison to the recovery of CO2 after door opening, the temperature fluctuation is less important.

Incubator temperature measurement DIN 12880

Incubator temperature measurement DIN 12880

Temperature range of incubators

In addition to the above, one should compare the necessary temperature difference between the surrounding lab and incubator temperature. Incubators usually only are able to heat but not to cool their chambers. For a precise regulation of the inside temperature, they have to be located in a room that has a lower temperature. The minimum temperature difference they can counteract usually ranges from 5-7 Kelvin in a good incubator. In labs without air conditioning, this can become a critical factor during the summer months as large windows and direct sun light onto the incubator can easily lead to local temperatures of 30°C and above. 

 

Principle of reaching 95% humidity

It is very important that no water evaporates from cell culture flasks, dishes or multi-well plates in the incubator. Otherwise the cells would be osmotically stressed or after some days even killed. Therefore, the humidity inside the incubator has to be maintained at about 95%. At the moment, there are three major ways in which this is achieved in cell culture incubators:

 

Direct water fill onto the bottom plate

cheaper than other variants
large surface for evaporation
water exchange is laborious and annoying
  (usually 1x per week)
cleaning has to be performed while the incubator is open (long door opening)
cleaning chemicals may evaporate and act on the cells
highest contamination risk
water has to be treated with additives to reduce germ growth
refill with autoclaved water

Incubator cell culture bottom water fill

Indirect water fill into tub on bottom plate

cheaper than the volatilization method below
medium surface for evaporation
much easier to clean than a bottom fill (1x per week)
tubs may be taken out for cleaning
cleaning outside the incubator allows
   free choice of disinfectant
medium risk of contamination
water has to be treated with additives to reduce germ growth
refill with autoclaved water

Incubator cell culture bottom water tub

Volatilization of water (steam injection)

safest variant
   as the water is decontaminated during volatilization
the reservoir may be hosting algae after some time
less cleaning effort (only reservoir),
   only water refills
do not add additives to reduce germ growth!
most expensive variant, higher costs for maintenance
cleaning of tubes and injectors not possible for lab personnel
humidity sensor needs maintenance,
   condensation should be checked

 

 

CO2 content and recovery after door openings

For the optimal growth of cells in an incubator, the CO2 content and its variation is the feature one should most look after. First, the incubator should keep the CO2 content as stable as possible and should recover very fast. Second, we should try to limit the loss of CO2 by keeping the number and duration of door openings as low as possible. For this, divided and well sealed doors as well as training of personnel are of crucial importance. Even though the DIN 12880:2007-05 standard does require the CO2 recovery measurement, one often has to inquire this separately from the manufacturer when comparing incubators.

Regulation of pH in cell culture media

The CO2 content of the incubator regulates the resulting pH in the used cell culture media. This means the CO2 content and the used medium are a system and work together to buffer the pH. When placing media in the incubator, an equilibrium between the sodium hydrogen carbonate solved in the medium and the CO2 content (in %) inside the incubator develops. The higher the CO2 content  in the incubator the lower the pH of a given cell culture medium. Normal CO2 contents are 5%, 8,4% or 10%, depending on type of medium used. During the culturing period, the cells continuously secrete acid waste products which make the medium become more and more acidic. The carbonate buffering system slows the acidification down but after 2-3 days its buffering capacity is reached, the color turns from orange to more yellow and the medium should be changed.

Phenole red pH cell culture medium

CO2 measurement types

 

The CO2 content of an incubator is measured by a special sensor. This sensor may be either a TC (thermal conductivity) or IR (Infrared) sensor. When choosing a new incubator, you should pay attention that the incubator has an IR sensor. TC sensors are influenced by the fluctuation of temperature and humidity resulting from the door openings. Therefore, the TC measurement is much slower and the true values for the CO2 content are not displayed in real-time. As a consequence, the display may already show a CO2 content of 5% but the true percentage is still lower. For the cells, this results in a longer phase of pH basification which adds up especially when the doors are opened successively. IR sensor react much faster as the CO2 content is measured directly and independently of humidity and temperature. They show true values in real-time, make the recovery faster and thereby regulate the medium pH more efficiently and precisely.

Another feature of the sensors is that some of them are built in such a way that they can remain in the incubator during a disinfection or sterilization routine. Other sensors have to be removed which takes more time and may be laborious.

Cell culture incubator sensor

Stability of CO2 content and recovery after door opening

 

The stability of the CO2 content and the recovery after door opening are usually measured according to the standard DIN 12880 and are given in % and min, respectively. In contrast to the temperature, the CO2 content is measured at a single central reading point. It is important to note that the standard was revised in 2007. The actual version is now DIN 12880:2007-05. The revision changed the time of the door openings (15s vs. 30s) and way the recovery data has to be analyzed (see temperature). As a consequence, measurements according to the new version lead to a longer recovery time. So, to compare incubators always inquire the time and measuring parameters.

Cell culture incubator CO2 display

Divided doors and reduced CO2 loss

With each door opening, CO2 and humid, warm air are lost from the incubator chamber. The larger the door and the longer the door remains open, the larger is the loss and the longer does recovery take. Therefore, the door openings are crucial factors influencing the stability of growth conditions for the cells. They may become a problem, when many people share one incubator and the doors are opened successively with high frequency (e.g. university labs). In order to limit the extent of loss it highly advisable to purchase an incubator with divided doors. Usually this results in a quite remarkable increase in price, but it's definitely worth it! You will later save money on CO2 and time on gas bottle exchange. In addition, the stability of surrounding conditions will make your results less variable. Always invest in divided doors! Depending on the manufacturer and model, incubators are offered with 2, 3, 4, 6 or 8 doors. As the doors of course change or limit the accessibility of the cell culture vessels inside, one should check the size and positioning of doors. For some incubators, 4 doors are a much better compromise than 8 viewed from both perspectives. In the foto on the right you see an old New Brunswick incubator which is now produced in a new and optimized design (see next section) by Eppendorf. Incubator divided doors

Leak tightness (sealing) of incubator doors

Another point where one can save money is the leak tightness of the door sealing and quality of the hinges of inner doors. Check the hinges for stability against vertical deformation and note where the doors are attached (only in the middle of the door is very instable). Compare the image on the right and the one above. The lower image shows the high quality door hinge of the new Eppendorf incubator. Also check whether the door closes well and whether its probable to not close the door correctly. Try to move the seals. Is it probable that they turn and create a leak ? 

For the outer door, check how the door is closed. Is it probable to not close the door be mistake ? Will it spring open ?

Cell culture incubator divided doors and hinges

Surface material of the inner incubator chamber

An important feature for the ease and efficiency of cleaning (DIN and GMP) is the chamber surface material. Three types are currently on the market:

Stainless steel (easy to clean as it has a very smooth surface, ATMP, TEMP as well as testing and production labs should choose this)
+/- Copper chamber and trays (bactericidal surface, but hard to clean as the corroded surface is very rough, expensive)
Copper/stainless steel alloy (supposedly also bactericidal but we have not seen data on this and no manufacturer send data so far, expensive, don't buy this unless you see prove)

Due to the cleaning efficiency and the prize the clear favorite is stainless steel for all labs in our opinion. Safe the money on copper and instead by divided doors!

 

Cleaning of incubator chambers (risk and effort)

This is a feature that initially is often ignored. However, efficient cleaning of the incubator is essential for contamination prevention and the easier this is achieved the smaller the risk of germ growing in the incubator. If we are honest, no one likes to clean the incubator. Especially not when racks and plates have to unscrewed, have corners and angles. So, the line of decision is clear - less is definitely more in this respect. When buying an incubator and when cleaning, think about two points:

  • in corners, clefts and holes, dirt and dust can form a basis for germ growth that is hard to reach while cleaning
  • cleaning and disinfection of inner racks, rods and ledgers are often not performed accurately because it is laborious and takes very long. Remember, sterile dirt is soon being repopulated after disinfection!
Incubator chamber design Binder

Ease of cleaning: what to look for ?

  • inner chamber without welding seams = drawn-out stainless steel (a MUST for ATMP and TEMP)
  • rounded corners
  • doors without welding seams
  • Lowest amount of inner rack parts, no shelves screwed to inner chamber, no ventilator
  • Easy disassembly of inner chamber parts for cleaning

In our opinion, the best inner construction design is available from Binder. The anti-plenum design is unbeaten as it has no inner parts except for the trays. The new Eppendorf incubators also have a inner design that is fairly easy and fast taken apart for cleaning.

 

Decontamination strategies inner chamber

Here, the state-of-the-art technology is by now an automated decontamination or even sterilization feature (sterile in place, SIP). Depending on the intended use of the incubator, one has to pay attention to the limits of these two hygiene measures as well as differences between manufacturers. Method, temperature and duration vary:

  • 160-180°C for 2 hours:
Heat sterilization according to or better than standards (ISO) and pharmacopoeias  (for ATMP and TEMP)
  • 120-159°C for 2 to 3 hours:
Decontamination but not sterilization (sufficient for research and assay labs)
  • Hydrogen peroxide sterilization:
So far, only available from Panasonic. H2O2 (Hydrogen peroxide) is a trend in pharma labs. Accordingly, it is in line with new developments that this decontamination method now appears for incubators and laminar flow cabinets. The major advantage is that the decontamination is rather fast (3h). However, afterwards all inner parts are wet and have to dry off.

 

Sterilization: Requirements according to standards and pharmacopoeias

The terms sterilization and decontamination are differently defined in international standards and pharmacopoeias in Europe, USA, Japan etc. Disinfection is a so-called germ reduction by 3-5 log units (1:1.000 - 1:1:100.000 bacteria would survive). Sterilization starts at a germ reduction of 6 log units and above (1:1.000.000 bacteria would survive). Depending on the intended use or process either disinfection or sterilization have to be ensured and validated.

Standard or Pharmacopoeia Temperature Duration
European Pharmacopoeia 160 °C 120 min
US Pharmacopoeia 170 °C 120 min

DIN EN 556
(Sterilization of medical devices)

160 °C
180 °C
120 min
  30 min
     

Additional Decontamination strategies (Air)

Several manufacturers offer incubators where all ingoing gas (air and CO2) is filtered via HEPA filters. Even though each opening of the incubator door allows non-sterile air to enter the incubator, this is an additional risk mitigation feature and therefore, positive. However, filters are not cheap and they have to be replaced which means additional control and maintenance. In many cases, the use of filters also makes a ventilator necessary. This might be a contra-productive risk of contamination and vibration.

In summary, we think that air filtration is a feature that should be taken into account for ATMP, TEMP and IVF labs if available without ventilator. All other labs, especially university labs definitely do not need filtration.

 

Sockets and cable ports in the inner chamber

Another add-on in some incubators is the possibility to order the incubator with sockets or cable ports. This is a big advantage when you plan to perform new assays with equipment placed in the incubator like impedance measurements for cytotoxicity or migration or life cell imaging (video microscopy) with small microscopes that fit in the incubator like the JuLI. If you don't have a socket or cable port, cables have to be squeezed through the inner and outer doors (image on the right). This results in CO2 leakage and produces gradients inside the incubator with in turn may influence your assays or the other cells growing in the incubator and increases your use of CO2 . Cable ports can be  sealed better than the doors and sockets circumvent the problem completely. Usually they are integrated in the chamber sides and have to be ordered with the incubator as they can not be installed later on. Not all manufacturers offer this feature. Binder does and additionally, you can also decide where you want it to be positioned (surcharge).

Cell culture incubator cable feed through

Vibrations (e.g. ventilators and pumps)

Ventilators and pumps may produce vibrations that might disturb your experiments or cell culture. They are true No-Gos for experiments like colony formation assaysstem cell and spheroid cultures. Additionally, they might influence adhesion of cells plated in flasks and multi well plates, producing unwanted patterns and distribution over the vessel surface.

This is the second reason -besides the contamination risk -, why we do not recommend incubators with ventilators. Additionally, the general standards for incubators does not demand any measurement in this respect. Therefore, one has to inquire this from the manufacturer if at all available.

 

Additional O2 control

Labs working on oxidative stress might want to consider the additional possibility to regulate oxygen by pumping nitrogen into the incubator. Many experiments over the last decade have shown, that the physiological oxygen partial pressure in tissue is way below the 21% in normal air (approx. 19-20% in the incubator). The trend to lower the oxygen content moves forward only slowly but this type of experimental setup will surely become more important in the near future in this field.

 

Qualification and validation: service support

In GMP and GLP labs, as well as labs producing ATMP and TEMP, all quality relevant equipment has to be qualified and methods have to be validated. This starts with the purchase of the equipment where according to the DbD concept (quality by design) a USR (user requirement specification) has to be compiled. The USR lists all features and their acceptable ranges that the user wants to ensure. The manufacturer of choice would then reply to the URS commenting the URS specifications and listing the individual ranges they can ensure. For larger installations, a FAT/SAT (factory or site acceptance test) has to be performed. When the incubator is installed it has to be qualified in the lab of use according to the VMP (validation master plan). The 4Q qualification method includes DQ, IQ, OQ und PQ (design qualification, installation qualification, operational qualification and performance qualification). It is rather laborious and lots of paper has to be produced. In order to limit the hassle for customers, many manufacturers now offer validation support. This can range from offering documentation and performance templates to on-site validation up OQ or even PQ phases. Prizes and extend of services differ greatly.

 

Manufacturer overview and where to order incubators

Below we are continuously collecting data for the comparison of incubator manufacturers and and overview of available features that are meant to help you decide which incubator is the best for you. In the very last section we have summarized a ranking of features for different types of labs. If you have additional comments or information, please contact us by mail or phone. User experience is always important!

 

Eppendorf (formerly New Brunswick incubators)

Eppendorf AG  
Production sites Germany
QMS ISO9001:2008
Ventilator + No.
Vibrations + No or little. No ventilator, no fan in air jacket.
Jacket

Water jacket without fan, but direct heating system of all 6 sides (includes door).

Door heating Yes.
Surfaces
Cell culture incubator drawn-out, rounded steel corners Eppendorf

Inner chamber completely drawn-out stainless steel, no welding seams. Small number of inner parts easy to disassemble for cleaning.

4 or optionally 8 trays.

Copper optional.

Number of inner doors
Cell culture Incubator Eppendorf divided doors and hinges
1, 2, 4, 8 (depending on model).
Door hinges Very stable and attached over the complete height of the doors in new models.
Temperature recovery Not data available.
Temperature range

4°C above RT up to 50°C.

Galaxy R and S with optional cooling system for labs without air conditioning.

Temperature stability Deviation from 37°C: +/- 0,1K measurement precision of control, +/- 0,3K homogeneity or uniformity over chamber.
Humidity control
Cell culture Incubator Eppendorf chamber layout

Removable tub (stainless steel).
Humidity: 95 %.

Humidity control optional.

CO2 range 0.2-20%.
CO2 recovery No data available.
CO2 stability Deviation from 37°C: +/- 0,1 % measurement precision of control, +/- 0,2 % homogeneity or uniformity over chamber.
Sterilization / Disinfection Automated disinfection routine sufficient for research and assay labs but below sterilization requirements for pharmacopoeias and standards for medical devices. Not yet required for ATMP and TEMP producing labs.
120°C, 240 min.
Decontamination duration Incl. heating, decontamination and cool down: 14h.
Sensors Infrared sensor for CO2, low in maintenance.
All sensors (except O2) are sterilizable and may remain in the incubator for the automated decontamination routine.
Sensor location Top-middle. Ensuring precise and realistic CO2 measurements.
HEPA-filter Yes, in the gas tubing.
Data logger 3-day data logger control via display and RS 232 interface for computer connection and real-time control.
Validation support IQ/OQ optional (surcharge).
Cable ports and sockets inside No.
Oxygen control Possible in some models.
Energy consumption 100W at 37°C.
Price range Medium to high.
Known advantages
  • Eppendorf Quality and reliability IR Sensor
  • Door heating
  • Divided doors optional, good hinges
  • Inner chamber drawn-out, little interior material, easy to disassemble, easy to clean
Known disadvantages
  • Decontamination at only 120°C
Additional Information We have worked with a Galaxy R incubator for several years now. We decided for this model as it has no incubator, a removable water tub and is really easy to clean. The data logger is very helpful to control how personnel uses the incubator and to trouble shoot. The doors of the first model were a little instable but Eppendorf has solved this in their well-known quality. Now the hinges are attached over the complete height of the doors and appear so far very very stable and tight. Due to the data logger and the very fast IR sensor we were able to monitor the true effects of door opening frequencies and duration. We use this quite often in our courses to demonstrate sources of experimental variation. The decontamination below GMP standard is not a problem for us as we are a research and training lab. Great machine, good support!
Web site Products

 

Binder

 
Production site Germany
QMS ISO9001:2008
Ventilator + No.
Vibrations No data available.
Jacket

Air jacket.

Door heating No data available.

Surfaces

Cell culture Incubator Binder chamber layout without rack

Inner chamber completely drawn-out stainless steal, no welding seams, rounded corners.

NO inner parts except trays = highest cleaning ease.

3 or optionally more trays.

 

Number of inner doors

Cell culture Incubator Binder divided doors

1, 4
Door hinges Stable but not over the complete height of the doors.
Temperature recovery Fast recovery: 4min (after 30s door opening; new DIN12880:2007-05)
Temperature range 5-7°C above RT up to 60°C
Temperature stability Deviation from 37°C: +/- 0,1K measurement precision of control, +/- 0,3K homogeneity or uniformity over chamber.

Humidity control

Cell culture Incubator Binder special water tub with condensation area

Removable tub (stainless steel) with special cool trap that limits condensation in the incubator to the water tub instead of walls or doors.

Humidity 90-95%.

CO2 supply Supply after homogeneous mixing with air via a special valve.
CO2 recovery

Fast: 7min (after 30s door opening, new DIN12880:2007-05)

Sterilization Automatic sterilization according to pharmacopoeias and standards for medical devices.
180°C, 120 min
Sterilization duration Incl. heating, decontamination and cool down: 10h.
Sensors Infrared sensor for CO2, low in maintenance.
All sensors are sterilizable and may remain in the incubator for the automated decontamination routine.
Sensor placement Top. Ensuring precise and realistic CO2 measurements.
HEPA-Filter No.
Data logger Data logger control via display and USB. Ethernet interface for connection to computer and real-time control using the Binder software Apt.com.
Validation support Yes. Full IQ/OQ support, trained personnel, documentation included. (Surcharge)

Cable ports and sockets

Cell culture Incubator Binder cable prot and feed-through

Cable ports are optional and may be ordered with special positioning (surcharge).
Oxygen Optional.
Energy consumption 100W.
Price niveau Middle to high.
Known advantages
  • Best inner chamber design (cleaning ease and speed)
  • Automated sterilization according to requirements
  • Excellent technical support during shipping and installation
  • Qualification and validation service
  • Lockable doors
Known disadvantages  
Additional information We have tested the CB160 some time ago and were very satisfied. An absolute surprise was the transport support. Specialized fright personnel only transports Binder incubators. They all receive special training on the incubators and do not only transport the incubators to the lab but also install them there. No stress, no worries, no: "only up to here and not further" - that was excellent!  The inner design of the chamber proved really well thought-through and functional. As promised we didn't see any condensation on doors or wall. All doors are stable and especially the outer door handle avoids that the incubator remains or springs open by accident.
Web site Products

 

Additional manufacturers of cell culture CO2 incubators

Hettich (Products; lanched in 2016), Memmert (Incubators), Panasonic (Incubators), Thermo Fischer (Incubators, Heraeus), Dunn Labortechnik (Incubators), NuAir distributed by IBS Integra Technomara (Incubators), Ewald Innovationstechnik (Incubators), Infors AG (Incubators), Labotec GmbH (Incubators), Selutec GmbH (Incubators).

 

Purchase decision criteria for research labs

The price, for sure, is a major factor in almost all labs. However, sometimes it makes sense to pay a little more initially instead of having trouble and problems afterwards. This is our ranking of criteria for labs outside the regulated field of production.

  • no ventilator unless it is easy to clean and disinfect
  • divided doors, good hinges
  • water tub
  • as little inner construction as possible, no welding seams
  • fast CO2 recovery after door opening
  • price
  • data logger without PC for maintenance and user control
  • heat disinfection automated

 

Purchase decision criteria for assay labs

Here, the main focus is consistency, reproducibility and low data variation. Additionally, most assay labs work according to ISO standards or under GMP/GLP. This is why we have a modified ranking of purchase criteria.

  • no ventilator unless it is easy to clean and disinfect
  • divided doors, good hinges
  • water tub or steam injection
  • data logger with PC for automatic control and documentation (validated)
  • as little inner construction as possible, no welding seams
  • cable ports if applicable
  • rather two small incubators than one large one
  • fast CO2 recovery after door opening
  • price
  • heat sterilization =/> 180°C for 2 h

 

Purchase decision criteria for  ATMP and TEMP labs

ATMP and TEMP are medical products and therefore, underlie strict control and have to be of high and reliable quality. They have to fulfill specification requirements and the technical equipment used in production has to be state-of-the-art technology. This is why some of the criteria here become a MUST:

  • An incubator which is manufactured according to ISO13485 (medical devices) would be a very strong pro for authorities (compare to risk analysis). However, at the moment there are not many incubators that fulfill this and hence, it is not mandatory
  • heat sterilization =/> 180°C for 2 h (State-of-the-art is required, SIP is preferred, sterilization is better than disinfection, not yet mandatory)
  • no copper surface (hard to clean, not allowed)
  • as little inner construction as possible, no welding seams (State-of-the-art is required)
  • data logger with PC for automatic control and documentation (validated) otherwise manual control and documentation has to be performed at least daily
  • Control and user management has to comply with ISO standards
  • rather two small incubators than one large one 
  • fast CO2 recovery after door opening (consistent quality, all deviations have to be monitored)
  • water tub or steam injection (the latter might be safer)
  • no ventilator unless it is easy to clean and disinfect