Incubator Sterility Test

By NuAire

Incubator contamination is a potential for all incubators. Laboratories have thousands of airborne contaminants that may enter a culture incubator during a door opening and enter the growth environment.

High Efficiency Particulate Air (HEPA) filters remove airborne contaminants. NuAire incorporates a HEPA large capacity capsule filter into the Autoflow CO2 Water-jacketed Incubators to remove contaminants that enter the chamber during door openings. The HEPA filter is incorporated into the recirculation system (See Figure 1). The chamber air is drawn into the inlet tube, to the pump, through a 0.3 micron HEPA inline capsule filter, through a 0.3 micron Hydrophobic HEPA filter to the IR Sensor and returned to the chamber. The recirculation provides filtration to remove airborne contaminants reducing contamination.

To test the incubator’s ability to filter the airborne contaminants, a biological test was developed.

Materials & Methods

A NuAire CO2 Water-Jacketed Incubator with setpoint parameters of 5% CO2 , 96% humidity and 37?C was tested. The incubator was set and stabilized for 24 hours. The incubator shelf placement was standard with four shelves equally spaced in the chamber. On the middle shelf, covered soy agar plates were placed on the center plane from the side access port (See Figure 2).

A nebulizer was used to deliver B. subtilis var. Niger spores prepared to a concentration of 1.0 x 104. The nebulizer was mounted next to the side access port to distribute the spores.

A wire hook was also present at the side access port to remove the agar plate covers.


A. Remove (2) control agar plate covers.

B. Place nebulizer over side access port. Connect air source and run nebulizer for one minute.

C. Remove nebulizer and agar plate covers per the following:

Plate 1 - 5 minutes
Plate 2 - 10 minutes
Plate 3 - 15 minutes
Plate 4 - 20 minutes
Plate 5 - 25 minutes
Plate 6 - 30 minutes

D. Allow agar plates to incubate for 24 hours. Remove agar plates and record.


Three test results indicated below. Each plate was analyzed for colony forming units (CFU) of the B. subtilis var. Niger spore.

Test #1:
Plate 1 - 122 CFU
Plate 2 - 69 CFU
Plate 3 - 27 CFU
Plate 4 - 19 CFU
Plate 5 - 16 CFU
Plate 6 - 4 CFU
Control Plates - TNTC

Test #2:
Plate 1 - 67 CFU
Plate 2 - 35 CFU
Plate 3 - 20 CFU
Plate 4 - 7 CFU
Plate 5 - 3 CFU
Plate 6 - 1 CFU
Control Plates – TNTC

Test #3:
Plate 1 - 200 CFU
Plate 2 - 150 CFU
Plate 3 - 75 CFU
Plate 4 - 16 CFU
Plate 5 - 7 CFU
Plate 6 - 1 CFU
Control Plates – TNTC
TNTC – Too numerous to count

Control plates were evaluated to check spore concentration was an acceptable challenge. The control plate should contain greater than 300 CFU’s to be valid. 


Results indicate a reduction in chamber spore concentration. The reduction of spores can be directly attributed to filtration through the HEPA filtered recirculation.

Results also indicate cleanliness level of class 100 or better 15 minutes after the chamber has been exposed to airborne contaminants. The filtration system reduces the chance for contamination.

The NuAire CO2 Incubators offer a substantial reduction in contamination potential.

Published In

Laboratory Etiquette Magazine Issue Cover
Laboratory Etiquette

Published: May 9, 2011

Cover Story

Laboratory Etiquette

Many lab managers still remember them from their student days—a handful of hastily stapled printouts sternly titled “Laboratory etiquette—Acceptable standards of conduct.” Those were rules to live by, and the smallest violation landed a budding laboratory scientist in front of the ticked-off chief instructor.
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