CO2 INCUBATORS

CONSTANT CONTAMINATION CONTROL

Nuaire’s Smart Designs and Concepts for Optimal Cell Growth
While minimizing Contamination

Introduction:
CO2 incubators play an essential role in any cell culture laboratory. Downtime of a lab’s
CO2 incubator can bring an entire lab’s work to a stop. The largest single reason for
incubator down time is contamination.

Contamination has been a major concern with CO2 incubators specifically and cell culture
laboratories in general for many decades. Many technologies have been developed to
eliminate contamination from various chemical disinfectants, filtration, heat sterilization
cycles to UV radiation to name a few. With regards to the cell culture CO2 incubator there
are several techniques being employed by different manufactures with varying degrees of
success. Currently the popular techniques to combat contamination are heat sterilization, UV
radiation, and the use of copper metal in the incubator inner chamber. While all 3 can be
effective against contamination if used correctly, misapplication of the technologies can
exacerbate the contamination problem.

It must be remembered that CO2 incubators provide ideal growing conditions for not only the
intended cells lines but also for any number or unwelcome bacteria, molds, yeasts, spores
and fungi. Therefore, not only must a CO2 incubator provide the ideal conditions for cell
growth, but in tandem, it must minimize or eliminate contaminants that can damage or destroy
cell lines. This dual goal can be achieved, but only with a TOTAL CO2 incubator concept,
and not by the simple add-on of a heat or UV radiation cycle to a traditional CO2 incubator.
ONLY SINGLE PIECES OF THE ANTI-CONTAMINATION PUZZLE

UV RADIATION

UV light has been used for many years to destroy contamination. UV light in the 253-254nm
range is most effective against microbes.

One example with which most any lab researcher will be familiar is the UV light inside of a
Laminar flow or Biological Safety cabinet (BSC). And it is common practice to leave the UV
light on over long periods of time, sometimes over night to produce any sterilizing effects.
Consequently, UV light use in a BSC is only a small contributor in the fight against
contamination. Depending on UV alone DOES NOT get the job done and lulls labs personnel
into a false sense of security. There is no substitute for good laboratory practice and
procedure, and this includes periodic wipe down and decontamination of the BSC, even if it
does have a UV light.

The same holds for true for a CO2 incubator. One simply cannot rely on UV alone to take
care of the problem. Below is a more detailed explanation as to why this is the case:

1) Exposure Necessary to Kill micro-organisms is purely a product of time and

intensity. Microbial inactivation depends on the UV-C dose that is described as UV
intensity multiplied by exposure time.

And UV light does not act quickly. Time is needed even with very high intensities for
microbes to be destroyed. In the best case scenario, 5-10 minutes of direct exposure to
UV light with an intensity of 40 microwatts per square centimeter is required to kill
most microbes.

Known data from CO2 incubators with UV light capabilities on the market today have
Maximum intensity in the 30 microwatts / sq. centimeter range. This intensity
represents almost point blank, direct exposure to the UV lamp. Furthermore, the
typical UV bulb used in CO2 incubators have not been more than a relatively weak 4
watts. Table 1-1 shows required UV doses to kill many common microbes:
 Bacteria *Dose Molds Dose Yeast Dose
Bacillus anthracis 8,700 Aspergillus 77,000 Common yeast 13,200
amsteldoma
B. Magatherium (veg.) 2,500 Aspergillus flavus 99,000 S. cerevisiae 13,200
B. Magatherium (Spore) 5,200 Aspergillus niger 330,000 S. ellipsoideus 13,200
B. Subtillus (spores) 22,000 Mucor Mucedo 77,000
Clostridium tetani 23,100 Penicillum digitatum 88,000
Clostridium botulinum 11,200 Rhizopus nigricans 220,000
Escherichia coli 6,600
Legionella pneumophila 12,300
Micrococcus Candidus 12,300
Mycobaterium tuberculi 10,000

Table 1-1 *Dose is microwatts / square cm.

Of interest in table 1-1 is the relative difficulty of killing molds, spores or any microbe of
larger size. Without direct and constant UV exposure larger micro-organisms go
comparatively unaffected. And by and large, most contamination in CO2 incubators tend to
be molds and spores introduced into the incubator during a door opening. Therefore, UV light
is not an effective tool against common, airborne CO2 incubator contamination.

Figure 1-1 makes this point even clearer on both levels; that is, 1) larger particles are harder to
kill with UV radiation and 2) this is particularly so when they are airborne as the microbes are
constantly in motion and never in one place long enough to receive enough exposure to the
UV lamp
Figure 1-1

*Figure1-1 shows the impact of a HVAC system

with UV radiation, with 25 microW/cm2, placed in a
recirculation loop. Spores are relatively unaffected
by the UV, but the viruses are markedly reduced.
This model incorporates chronic dosing effects
from recirculation with an exposure of 0.2 seconds
for each pass.

Source - “Airborne Respiratory Diseases and Mechanical Systems for CONTROL OF MICROBES”,W.J.
Kowalski, William Bahnfleth, Pennsylvania State University Architectural Engineering Department
 Figure 1-2 Relative sizes of various microbes

2) Performance of UV lamps are affected by many factors including temperature, air

flows, dirt/dust, humidity, time and available reflective surfaces.
a. UV bulbs operate most efficiently at room temperature 70°- 80°F (20° - 25°C).
Temperatures either higher or lower than this optimum value decrease
effectiveness of the bulb. The same holds true for humidity. High humidity %
will adversely affect the UV bulb.
b. Microbes that are attached to larger particles such as dust have a much greater
chance of avoiding direct exposure to the UV radiation.

Reminder:
Room air being introduced into the incubator is the single largest
Source of Contamination.

Common room air is full of dust particles on which microbes can attach. In
addition since dust or dirt is not collected as it would by filtration, a build up
over time of these larger particles is likely in the inner chamber and on the UV
bulb itself reducing bulb intensity and surface reflectance.
c. UV bulbs lose intensity over time. Lost intensity translates directly into
decreased kill rate as time progresses.
d. Airflows hinder UV performance is a couple of ways.
1) If airflow is moving quickly as would be the case in an air circulating
duct there is not much exposure time to the UV. In order to offset the
short exposure time, multiple, high wattage UV lamps strategically
placed in the air duct are needed to do an effective job.

Reminder:
Today’s CO2 incubators with UV light capability use only
one 4 watt UV bulb.
 2) If air is moving slowly (as is the case with current CO2 incubators
with UV light capability) chances are high that all of the chamber air
is not passing within exposure range of the UV lamp leaving many
airborne microbes unaffected.
e. Reflectance of UV light is critical in an enclosed space, especially a space that
has a square shape. Aluminum has proved to be the best UV reflecting
material with a reflectance of up to 85%. Stainless steel and copper, common
inner chamber material for CO2 incubators, in contrast reflect 30% or less.

Copper or Copper Enriched Stainless Steel Inner Chamber:

Copper has been known for millennia to have anti-microbial properties. However, in order
for the anti-microbial properties to manifest themselves, the copper must come into direct
contact with water. Copper fungicides/bactericides can be described as insoluble compounds
yet their anti-microbial action is precisely due to the release of minute quantities of Copper
ions when in contact with water. By just putting water on the surface of Copper, quantities
of Copper ions are in the 1 ppm (parts per million) range. 1 ppm is toxic enough to kill many
microbes but others are more resistant needing over 3-5 ppm to be killed. Therefore, without
water being present, copper is not a potent threat in any way to microbes. And even with
water present there is the potential for some microbes to survive because Copper ion toxicity
is not high enough.

Several brands of incubators have used copper or copper enriched stainless steel for the inner
chamber of the CO2 incubator. In a CO2 incubator, condensation would be the only water
source that might come into contact with the inner chamber. And most every incubator built
today is specifically designed to avoid any condensation precisely because any microbial
growth must have water to start. Consequently without condensation forming on the inner
chamber surfaces, copper or copper enriched stainless steel will play little if any role in the
fight against CO2 incubator contamination.

But assuming for a moment that the inner chamber walls and shelves were wet, the copper
ions responsible for the anti-microbial effect would be consumed or metabolized and
consequently the copper’s effect on the microbes would diminish over time. Unless new
particles are exposed by some sort of metal removal process like sanding down the inner
chamber, the anti-microbial benefits would slowly disappear.

Because of Copper’s unique microbe fighting ability its most prevalent use in this manner is
in agriculture and not in the laboratory:
SPECTRUM OF FUNGICIDE ACTION

*Table 1-2

SPECTRUM OF BACTERICIDE ACTION

**Table 1-3
*/** Source –Industrias Quimicas del Valles, s.a., Copper information Guide
Heat Cycle Decontamination Routines:

Heating the inner chamber of the incubator to elevated temperatures, is another common
method to fight contamination. This technology has been directly borrowed from
Autoclaves/Sterilizers and has been adapted for use in CO2 incubators. And it has been
proved effective in many studies for eliminating most all forms of microbes from all internal
surfaces and components in a CO2 incubator

Both moist heat (steam heat) as low as 90°C and dry heat at or above 140°C have been
independently proven to kill most any mircoorganism on the incubator’s inner chamber
surfaces although there is still on-going debate about which type of heat, moist or dry is better
for killing specific types of microbes.

However, heat decontamination cycles are only effective in killing microbes at one given
point in time on the inner chamber surfaces of the incubator. As soon as the incubator door is
opened and the inner chamber is exposed to room air, the incubator is once again
“contaminated”.

Reminder:
Room air being introduced into the incubator is the single largest
Source of Incubator Contamination.

Heat decontamination cycles can therefore only be thought of as a temporary fix to on-going
contamination worries. Once room air is re-introduced into the incubator after the heat decon
cycle is completed, sterility is lost.
 NUAIRE’S TOTAL INCUBATOR CONCEPT
COMPLETES THE ANTI-CONTAMINATION PUZZLE

CONSTANT CONTAMINATION CONTROL

Rather than think about contamination as an after-thought or as something that will inevitably
happen in all CO2 incubators, Nuaire chose to be more proactive and keep contamination
from happening in the first place. Nuaire’s incubator design and philosophy are simple yet
effective:

1) Clutter Free Interior

2) Closed loop HEPA filtration under slight positive pressure – Positive Sterile Air Flow

And as a complimentary addition to Nuaire’s proactive approach to avoiding contamination

before it has the opportunity to take hold, we have also developed the most unique, easy to
use and effective dual heat decontamination cycle on the market today.

3) Dual Heat Decontamination Cycle

a. 95°C Wet (Humidified Cycle)
b. 145°C Dry Cycle

Once again, heat cycles are only good for one point in time, but they do make sense in certain
instances such as 1) after one line of research is complete and before new research is started
or 2) work done in the incubator could be potentially harmful to people – research on HIV for
example.
Clutter Free Interior

Nuaire has manufactured essentially an empty inner chamber. The objective is to make the
incubator easy to wipe down with a disinfectant when required and easy to
disassemble/reassemble the shelves and supports (no tools required).

In order to create an empty chamber there is minimal hardware such as screws, sidewall
plenums, fan wheel, and filter, etc. Any items that can be eliminated have been and those that
could not ( pump for air circulation, HEPA filters, IR sensor) have been remotely placed
exterior to the inner chamber.

Image 1-2
Easy to remove shelves and supports
Image 1-1
Empty / Clutter Free Chamber

Image 1-3 Image 1-4 Image 1-5

Single Piece / Removable / Autoclavable Rounded Corner Interior for Inner Door Handle
Vulcanized Silicone Gasket easy cleaning No Door Handle hardware extends into chamber

All interior parts are easily removable and autoclavable. Once removed surface
decontamination is quick and easy.
HEPA Filtration
Nuaire is globally recognized as a leader in HEPA filtration and airflow from our Class II
Biological Safety and Laminar Flow Cabinets. This expertise has been has been applied and
adapted to the Nuaire line of CO2 incubators. Just as HEPA filtration has been proven safe
and effective in BSC’s for trapping and containing small particles down to 0.3 micron or
smaller, the same holds true for incubators if the technology is properly applied.

Typical laboratories have thousands of airborne contaminants that may enter an incubator
during a door opening, and of course the contaminants find the perfect environment in which
to thrive and reproduce. Elimination of the airborne contaminants as they enter the incubator
would be the ideal solution, but this is not feasible given today’s technology. However
Nuaire’s innovative chamber design, maintaining a slight positive pressure within the
chamber and by applying our expertise in High Efficiency Particulate Air (HEPA) filters,
Nuaire has greatly reduced the chances of sample contamination and have virtually stopped
contamination before it can start.

Nuaire incorporates a HEPA filter into both the water jacketed and direct heat model
incubators. The HEPA filter is incorporated into the recirculation system and removes
airborne contaminants that enter the chamber during a door opening. See Figure 1-3
Figure 1-3

Air within the incubator is constantly recirculated through the HEPA filter, and with the
positive pressure of the inner chamber, chamber air tends to fall out of the incubator rather
than room air being drawn in. The positive pressure therefore minimizes the amount of room
air that is able to enter the incubator during a door opening.
In contrast, other brands of incubators that have attempted to use a HEPA filter and even
those brands without HEPA filtration keep the inner chamber under negative pressure and the
blower wheel does not turn off when the door is opened. The result is large of amounts of
room air and contaminants are drawn into the incubator. See figure 1-4

Figure 1-4

Negative Negative

Even with a HEPA filter with this type of airflow, contamination has a good chance of taking
hold because of the amount of room air that is being drawn into the chamber and because of
the fact that the HEPA filter is directly inside the chamber and exposed to the cell lines. Any
variation of the negative pressure air flow and contaminants that are airborne or that are
trapped on the outside of the filter media can and will fall down onto cell lines.

To test Nuaire incubators’ ability to filter airborne contaminants a biological test was
developed

A Nuaire incubator with set point parameters Figure 1-5

of 37°c, 5% CO2 and 96% humidity was tested.
The incubator was stabilized for 24 hours. The
incubator shelf placement was kept standard with
four shelves equally spaced in the chamber, without
causing interference to the side access port.

On the middle shelf, covered soy agar plates were placed

on the center plane from the side access port.
See figure 1-5.
A 316 Upgrade Stainless Steel 6 Jet Figure 1-6
Collision Refluxing Nebulizer was used
To deliver B. Subtilus 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 B. Subtilis
spore suspension into the incubator chamber. See figure
1-6. A wire hook was also present at the side access port
to remove the agar plate covers during the test. Once all the
above materials were in place, the following procedure was
initiated.

PROCEDURE

A) Remove the (2) control agar plate covers via side access port
B) Place nebulizer nozzle through side access port, connect
20 PSIG air source and run nebulizer for one minute.
C) Remove nebulizer and remove agar plate covers per the following schedule:

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 the agar plates to incubate for 24 hours
E) Three replicate tests be performed

RESULTS

Test 1 Test 2 Test 3

Plates 1 -122 CFU Plates 1 - 67 CFU Plates 1 - 200 CFU
Plates 2 - 69 CFU Plates 2 - 35 CFU Plates 2 - 150 CFU
Plates 3 - 27 CFU Plates 3 - 20 CFU Plates 3 - 75 CFU
Plates 4 - 19 CFU Plates 4-7 CFU Plates 4 - 16 CFU
Plates 5 - 16 CFU Plates 5-3 CFU Plates 5 - 7 CFU
Plates 6 - 4 CFU Plates 6-1 CFU Plates 6 - 1 CFU
• CFU = Colony Forming Unit of B. Subtilis var. Niger Spore
• Control Plate CFU were too numerous to count
CONCLUSION

The testing indicates a substantial reduction in the chamber spore concentration. And the
reduction of spores is directly attributable to the HEPA filter recirculation system that
includes the 0.3 micron HEPA inline capsule filter. See Image 1-6.

The Results show a general cleanliness level of ISO Classs 5

(Formerly Class 100). Sterility is achieved in the water jacketed
incubators in approximately 15 minutes while same is achieved in
the direct heat incubators in 3 minutes. The difference is due to
different rates of air recirculation within the units. Having a
filtration system this effective will greatly minimize contamination’s
ability to grow. However, Good Laboratory Practice including the
the periodic surface disinfection of the incubator chamber is also Image 1-6
further reduce chances of contamination.

Additionally, adding a trace amount of copper sulphate to the water humidification pan will
eliminate contamination here as well. If a water pan is not desired at all, the other option is to
choose an incubator that has a humidity injection system that is both controlled and displayed
via the front control panel. The NU 4850 from the Nuaire incubator range serves this
purpose. See image 1-7.
Image 1-7
In the NU 4850, the water source is located
Exterior to the inner chamber. Water is taken
From the reservoir, heated and then steam
Injected into the chamber to obtain and maintain
Desired Humidity %.
Dual Heat Decontamination Cycles

The last piece of the puzzle is Nuaire’s unique dual heat decontamination cycles in the
NU 5510 incubator. As mentioned above, periodic decontamination of all interior
components and surfaces is necessary in certain situations.

145°c Dry Heat Cycle

Figure 1-7

145 DEG C DECON CYCLE SKIN TEMPERATURES

ROOM AMBIENT 20 DEG C

150
145
140
135
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30

TIME DURATION (HR:MIN:SEC)

The entire duration of the 145c cycle averages 10 hours. This includes warm-up, sterilization
period, and cool-down. The actual duration of the sterilization period is 3 hours.
95°C Humidified Cycle
Figure 1-8

100
95
90
85
80
75
70
65 RH
60 95 DEG. C T
55
50 HUMIDIFIED
45 DECON CYCLE
40
35
30
25
20
15
10
5
0

TIME (HR:MN:SEC)

Total duration of the 95c humidified cycle averages 14 hours. This includes warm-up,
sterilization period, and cool-down. Humidity is attained by filling the water pan with 300ml
of distilled water.

The dual heat decontamination cycle comes standard on the NU 5510 incubator. This gives
the choice to the user as to which cycle to run at any given time. Both Cycles are short
enough to be run over night meaning no downtime is caused.

Lastly, it is important to note that that NO INTERNAL COMPONENTS need to be

removed such as IR sensor, blower wheel, HEPA filter etc before running one of the heat
decontamination cycles. Not having to remove and re-install components minimizes
reintroduction of contaminants by eliminating unnecessary door openings.
FINAL COMMENTS

Nuaire’s line of CO2 incubators optimizes cell growth by providing ideal growing conditions
by using the latest in technology such as digital IR sensors, and humidity display and control.
And at the same time, contamination is virtually eliminated due to having one overall concept
aimed at avoiding contamination. Only Nuaire offers a clutter free, rounded corner interior,
closed loop HEPA filtration system under positive pressure, and the dual heat
decontamination cycles. It is no wonder therefore why Nuaire’s incubator contamination rate
is 0.003%. With more than 20,000 incubators sold there have only 60 documented
contamination cases that were not caused by user neglect or lack of maintenance.

Nuaire truly offers CONSTANT CONTAMINATION CONTROL.