Co2 Incubator

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CO2 INCUBATORSCONSTANT CONTAMINATION CONTROLNuaires 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 labs CO2 incubator can bring an entire labs 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 PUZZLEUV 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:

BacteriaBacillus anthracis B. Magatherium (veg.) B. Magatherium (Spore) B. Subtillus (spores) Clostridium tetani Clostridium botulinum Escherichia coli Legionella pneumophila Micrococcus Candidus Mycobaterium tuberculi Table 1-1

*Dose8,700 2,500 5,200 22,000 23,100 11,200 6,600 12,300 12,300 10,000

MoldsAspergillus amsteldoma Aspergillus flavus Aspergillus niger Mucor Mucedo Penicillum digitatum Rhizopus nigricans

Dose77,000 99,000 330,000 77,000 88,000 220,000

YeastCommon yeast S. cerevisiae S. ellipsoideus

Dose13,200 13,200 13,200

*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 lampFigure 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- 80F (20 - 25C). 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:Todays 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 coppers 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 Coppers 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 bor