Laboratory Incubator: Principle, Parts, Types, Uses, and Why Cultures Fail

Imagine coming in on Monday to read your throat swab cultures and finding the chocolate agar plates blank, no growth at all, even on the positive control. The plates were fine. The CO₂ incubator's gas cylinder had emptied over the weekend, and without the 5–10% CO₂ that fastidious organisms like Streptococcus pneumoniae and Neisseria species need, nothing grew. The incubator held 37°C perfectly the whole time. This is the lesson that runs through this entire article: an incubator that shows the right number on its display is not the same as an incubator giving your organisms what they need to grow.

A laboratory incubator is an equipment that provides a controlled environment for the growth of microorganisms. Whereas incubation is the process of providing a controlled environment.

Figure: Laboratory Incubator

Principle of Laboratory Incubator

A laboratory incubator works on the principle of thermostatically controlled heating. An electrical heating element warms the insulated chamber, and a thermostat linked to a temperature sensor switches the heater on and off to hold a set temperature. When the chamber cools below the set point, the sensor signals the controller to power the heater; when it reaches the set point, the heater switches off. A fan circulates the warm air so the temperature stays even throughout the chamber. This continuous feedback keeps the interior at a stable, preset temperature regardless of small changes in room temperature.

A predetermined temperature is set in the instrument because different microorganisms require different temperatures for growth. For example, the suitable incubation temperature for most human bacterial pathogens is 35–37°C, whereas for molds and yeasts it is 25–30°C. The temperature inside the incubator increases when power is supplied. The temperature control depends on the temperature sensor, controller, and contactor.

Once power is supplied, the contactor is energized and current flows to the heating element. Fans ensure even distribution of warm air throughout the chamber. When the incubator reaches the set temperature, the sensor signals the controller and the contactor is de-energized, cutting power to the heater until the temperature drops again. A thermometer displays the temperature inside the chamber.

Incubation Temperatures for Different Organisms

The single most common exam question and bench mistake is which temperature to set. The default of 35–37°C exists because most organisms that infect humans grow best at human body temperature. Organisms adapted to other environments need different settings.

Parts of Laboratory Incubator

The laboratory incubatory has the following parts:

Figure: Parts of a laboratory incubator

Cabinet with Door

The cabinet of the incubator is double-walled. The inner chamber is made of stainless steel, which resists corrosion and is easy to clean, while the outer body is made of steel with a coated finish. The glass wool insulates the gap between the double-wall to prevent heat loss. The storage capacity of the cabinet ranges from 20 liters to 800 liters.

The door is present at the front part of the incubator. The door has glass for visualizing the contents present inside the cabinet. Silicone rubber door gaskets provide an airtight seal between the door and the cabinet. The gaskets prevent hot air from escaping outside the cabinet and non-sterile air from entering the cabinet.

Note: Unlike a hot air oven, an incubator usually has a glass inner door so you can view the contents without breaking the temperature seal.

Control panel

The control panel is outside the cabinet and has many switches to control different parameters of the incubator. It also controls the thermostat.

Thermostat

The thermostat provides the desired temperature and maintains the temperature during heating and non-heating cycles.

Thermometer

The thermometer is in the top part of the outer wall of the cabinet. One end of the thermometer has gradations indicating the temperature, and another has a mercury bulb embedded inside the incubator.

Shelves

The shelves are present in the inner part of the incubator. These are removable, which makes their cleaning easy. The shelves hold the Petri plates containing culture media. Perforations on the shelves help circulate hot air throughout the incubatory.

Additional parts

Some incubator has HEPA filters which creates a closed-loop system to reduce the chances of contamination. Similarly, some incubators have humidity and CO₂ control to provide the desired environment for some microorganisms’ growth.

Types of Laboratory Incubator

Based on their size and purpose, the laboratory incubator is of the following types:

Cooled/Refrigerated Incubator

The cooled or refrigerated incubator has a refrigeration system in them. These provide temperatures below the ambient temperature. The cooled incubators offer precise temperature control and have air circulation fans that provide fresh air inside the chamber. It also has different sensors which monitor the temperature. These are used in biology and microbiology laboratory.

BOD (Biological Oxygen Demand) Incubator

BOD (Biochemical Oxygen Demand) incubators combine heating and cooling to hold low, stable temperatures, typically around 20°C. They are used for BOD determination in water and wastewater testing, and for growing organisms that prefer lower temperatures, such as environmental bacteria and some fungi.

Humidity Incubator

A humidity incubator is a special type of incubator with control settings for humidity. It is important because the growth of certain organisms requires some moisture. Temperature and humidity are controlled simultaneously. Indirect heating helps in maintaining the special atmosphere inside the cabinet.

CO₂ (Carbon Dioxide) Incubator

Some fastidious organisms need an atmosphere enriched with carbon dioxide (usually 5–10%) rather than ordinary air. A CO₂ incubator maintains this by injecting CO₂ from an external cylinder and holding it at a set concentration.

Commonly infrared or thermal conductivity sensors hold the CO₂ level. It also has a water tray for maintaining humidity levels. The incubator mimics the condition of the human body that makes it susceptible to contamination. Therefore, there is a special sterilization system that cleans the incubation chamber and prevents any contamination.

Shaking Incubator

A combined form of motor/shaker and incubator is termed a shaking incubator. The incubator provides controlled temperature, humidity, oxygen level, etc. It also offers constant shaking for a homogenous mixture of the samples like a centrifuge. It is mainly used in molecular biology and genetics.

Figure: Shaking incubator

Benchtop/Standard Incubator

Figure: Benchtop Incubator

Benchtop/standard incubators are the incubator present in the most common laboratory. The temperature ranges from ambient to up to 100℃. Most of these incubators have a glass door, alarms, and a display screen that displays the temperature and time.

Uses of Laboratory Incubator

The incubator has a wide range of use. Some of the applications are as follows:

• In microbiology laboratories, an incubator helps to grow bacteria, fungi, and other microorganisms.
• Similarly, it maintains cell cultures at body temperature, which is required for growing viruses inside those host cells.
• Likewise, in zoology laboratories incubator provides the environment for hatching the eggs of insects, birds, and other oviparous organisms.
• It also helps to store biological specimens before analysis in medical laboratories.

Why Cultures Fail in the Incubator

An incubator that displays the correct temperature can still ruin a culture. These are the failures that actually happen at the bench and how to prevent them.

1. The CO₂ cylinder runs empty. Capnophilic organisms like S. pneumoniae and Neisseria species stop growing the moment CO₂ drops, even though the temperature stays perfect. Check the cylinder pressure gauge daily and keep a backup cylinder connected through a changeover valve so gas supply never lapses over a weekend.

2. The door is opened too often, or left ajar. Every door opening dumps warm air and, in a CO₂ incubator, the gas atmosphere. Recovery takes many minutes. A door not fully latched, often because of a worn gasket, causes a slow constant leak that the display may not reveal until cultures fail. Open the door as little as possible and inspect the gasket seal regularly.

3. Overcrowding blocks airflow. Plates and flasks stacked against the fan or packed tightly create cold and hot spots. Colonies grow unevenly or slowly. Leave space around the internal fan and between stacks so warm air circulates freely.

4. Wrong temperature for the organism. Setting 37°C for a fungal culture that needs 25–30°C, or forgetting to move a Campylobacter plate to 42°C, gives a false negative. Confirm the required temperature before loading, and label incubators clearly when the lab runs more than one setting.

5. Plates and tubes dry out. Long incubations, especially at higher temperatures, dry the medium and kill the culture. For extended incubation, hold plates in a moist chamber with damp sterile cotton wool, and plug tubes with cotton to slow evaporation.

6. Condensation and contamination. Water pooling on lids drips onto colonies and spreads growth; the warm, humid CO₂ incubator is especially prone to fungal and bacterial contamination. Invert plates (agar side up), keep the water tray clean, and run the incubator's sterilization cycle on schedule.

7. Calibration drift. The display can read 37°C while the chamber sits at 35°C or 39°C after months of use. This quietly skews results, especially for temperature-sensitive organisms. Verify against an independent reference thermometer periodically and recalibrate.

8. Power interruption. In settings with unstable mains supply, an overnight outage cools the chamber and can kill slow-growing cultures. Where possible, connect critical incubators to a backup supply and check the temperature first thing after any known outage.

Precautions

While operating the incubator, you should follow the following precautionary measure:

• Avoid opening the door of the incubator unnecessarily.
• Also, maintain the temperature of the incubator carefully.
• Clean the incubator regularly.
• If incubation of Petri plates is done for a longer time, the Petri plates should be placed in a moist chamber with damp sterile cotton wool at the bottom.
• Similarly, for the incubation of test tubes for a long time and at a higher temperature, push a cotton plug inside the neck of the tube to prevent drying of the medium.

How to Remember

The display number lies, the atmosphere tells the truth. Temperature is only one of the three things an incubator controls. Before trusting any incubation, check all three: temperature, gas (CO₂ where needed), and humidity. Cultures fail far more often from the second and third than from the first.

"Body bugs grow at body temperature." The 35–37°C default is not arbitrary; it is human body temperature, and human pathogens are adapted to it. Any organism that wants a different temperature is telling you it lives somewhere other than the human body: fungi at 25–30°C (cooler environment), Campylobacter at 42°C (bird gut), Listeria at 4°C (soil and refrigerators).

Key Exam Facts in One Table

Where Students Get Confused

Incubator vs. hot air oven. Both are heated cabinets, but their jobs are opposite. An incubator holds a low temperature (around 37°C) to grow organisms. A hot air oven reaches 160–180°C to kill them (sterilization by dry heat). If a question mentions growth, it is an incubator; if it mentions sterilization, it is an oven.

CO₂ incubator vs. anaerobic jar. A CO₂ incubator provides an atmosphere with extra carbon dioxide but still containing oxygen; it is for capnophiles like S. pneumoniae. An anaerobic jar removes oxygen entirely; it is for anaerobes like Clostridium and Bacteroides. Capnophilic is not the same as anaerobic.

Thermostat vs. thermometer. The thermostat controls the temperature (it switches the heater). The thermometer only displays it. A working thermometer with a drifting thermostat still gives the wrong temperature.

"Cooled incubator" is still an incubator. A refrigerated or BOD incubator confuses students because it cools rather than only heats. It is classified as an incubator because it holds a precise set temperature for growth, just below room temperature rather than above it.

References

• Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
• Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2 (2nd ed.). Cambridge University Press.
• Manandhar, S., & Sharma, S. (2017). Practical Approach to Microbiology (3rd ed., pp. 14–15). National Book Centre.
• Vandepitte, J., Verhaegen, J., Engbaek, K., Rohner, P., Piot, P., & Heuck, C. C. (2003). Basic Laboratory Procedures in Clinical Bacteriology (2nd ed.). World Health Organization.