TEST REPORT ON

via ultraviolet radiation and ozone.

Summary

The tested UV/ozone sterilization unit from the company

As the contamination of cultures is directly dependent on the germ level in the incubator and the incubation time, the regular reduction in germs also substantially reduces in infestation of cultures.

Introduction

When carrying out biological work, the contamination (infestation with bacteria and fungus) of cultures represents a disruptive factor which necessitates the repetition of complex operations and may also result in the loss of expensive, non-reproducible parent cultures.

Established processes for degerming (purifying) cultures often fail to produce the desired success or involve unacceptable side effects.

As contamination can be caused by a large number of parameters, it is a logical step to restrict or eliminate the risks of contamination.

Incubation in CO2 incubators involves a high risk of contamination.

As a result of the required temperature and the high level of humidity, ideal conditions for growth prevail here, not only for the cultures but also for the fungus and bacteria.

However carefully the work is carried out under sterile conditions, contamination of these incubators is unavoidable, as air-borne germs enter the incubator when it is opened.

Infestation of the cultures with these germs cannot be completely prevented by the use of antibiotics and fungicides.

This contamination is effected by the conditions to which the cultures are subjected during >handling< and the level of germ content in the laboratory atmosphere.

Infestation of the cultures does not commence immediately upon contamination of incubator, but is determined by the type of culture containers employed and the time factor.

As the time factor (incubation time) is usually fixed, the risk of the cultures becoming contaminated is >from a purely statistical point of view< directly dependent on the level of germ content in the incubator.

It is therefore necessary to reduce the level of germ content in the incubator.

Previous decontamination and germ-reducing processes

 Disinfection by wiping

The standard agents available on the market are either limited in their effects or cytotoxic.

These agents may become deposited in joints and grooves, for example, and may also be absorbed by plastics and released on a gradual basis.

Inaccessible points remain untreated.

Disinfection by wiping requires very extensive and time-consuming work and must be carried out on a regular basis,

1-3 times per month.

 Copper salts

Although copper salts do prevent microbic contamination in the humidifying water, they also corrode metals, as a result of which glass or plastic dishes have to be used.

Microbic contamination of the interior is unavoidable.

Similar conditions apply when other fungicides or bactericides agents are employed as water additives.

Ultraviolet radiators

UV radiators are often employed in conjunction with disinfection by wiping. Degermation is possible only in the irradiated zones.

Hot-air sterilization

Hot-air sterilization at 80-180 ° C is possible for some

CO2 incubators. However, this method of sterilization is only optimal and effective at 180 ° C. These units are very expensive, though.

The combined UV/ozone sterilization process

 Description of equipment

The UV/ozone sterilization system comprises the separate control unit and the actual sterilization unit with the UV/ozone radiators, which is >connected via a flat cable< to the control unit.

The control unit contains all the electrical components required for the system to function, such as relays, safety switches, timer for setting the decontamination and ozone degradation time and indicator lights to show the various operational conditions.

The flat cable, which connects the control unit to the sterilization unit, can be clamped easily between the door seal, see Fig. 1.

The sterilization unit consists of a base for the installation of three special ultraviolet radiators with an output of

15 watts each, which produce ozone in the wave range < 200 nm.

After completion of the decontamination period, the control unit switches automatically to ozone degradation.

Located inside the sterilization unit are a platinum/palladium catalyst and a fan.

The ozone atmosphere is blown over the catalyst by a fan and the ozone is degraded.

Ozone measurement

As ozone is a highly toxic gas (threshold limit value at place of work = 0,1 ppm), the ozone concentrations in the room (during ozone treatment) and in the incubator (after completed ozone degradation) were measured for safety reasons >relating to staff and cell cultures<.

After approx. 15 min approx. 50 ppm of ozone was measured inside the incubator. After approx. 30 min, i.e. after ozone stabilisation, approx. 60 ppm of ozone was measured continuously, throughout the entire decontamination phase, see Fig. 2.

After the one-hour period of ozone degradation, the following concentrations of residual ozone gas were measured with the door closed:

• middle of interior maximum 0,05 ppm
• directly above the water surface
   maximum 0,07 ppm

After opening the door, i.e. after an exchange of air had taken place, no ozone was detectable inside the incubator or above the water surface.

No ozone concentration greater than 0,02 ppm was measured outside the incubator >including directly on the cable bushing< at any time during the decontamination period.

The threshold limit value at place of work for ozone is 0,1 ppm.

All ozone measurements were carried out with "Dräger measuring tubes"

type 10 A (measuring range: 10-300 ppm) and
type 0,05 B (measuring range 0,05-1,4 ppm).

 Purpose:

The purpose of the test was to analyse the germ count for air-borne germs (fungus) and the spreading and multiplying behavior in a UV/ozone-treated and an untreated CO₂ incubator.

Test configuration

For this purpose, we used the naturally occurring germs in laboratory atmosphere. In accordance with realistic handling practices, the CO₂ incubators were opened 5 times a day for 1 minute

Constant conditions were maintained in the two CO₂ incubators with an internal volume of approx. 200 litres each, with the temperature at 37 ° C, the CO₂ content at

5 % and the humidity at 95%

The following germ count indication means were employed for each incubator:

• 12 dishes containing nutrient agar, distributed evenly over three levels in the incubator.

• 1 impression preperation each from the two side walls and from the rear wall.

• 3 smears from inaccessible points which not covered by the UV radiation.

• direct germ count assessment in the humidifying water

• (5 Petri dishes per assessment).

Procedure

After carrying out basic cleaning of the two incubators and supplying them with aseptic, demineralised water, the test incubator was subjected to additional decontamination using the UV/ozone sterilization unit.

• Decontamination time: 2 hours
• Ozone degradation time: 1 hour

A germ count assessment was then carried out once again on the UV/ozone-treated test incubator, 1, see Table 1.

On all subsequent days during the testing period (42 days), the germ count assessment for the test incubator was carried out both before and after UV/ozone decontamination, see Table 1.

 Evaluation/Results

After disinfection by wiping, the water in both incubators was slightly contaminated and contamination could be determined on the metal surface inside the incubator. After additional UV/ozone-treatment, this contamination disappeared.

 The UV/ozone-treated test incubator was sterile, see Table 1, day 1.

"Fungus, bacteria and germs are destroyed by the UV/ozone-treatment"

The relatively low level of fungal contamination in the untreated incubator, 2, is due to the fact that this unit was consciously not operated under realistic practice, visible fungal contamination is usually detectable in a substantially shorter period of time.

The fungal contamination in the water of the untreated incubator rose in relation to the time factor up to the 21st day, after which stagnation was to be observed up to the 42nd day.

In the case of the UV/ozone-treated incubator, fungal contamination in the water was not established until the 42nd day; this contamination was again completely suppressed after the decontamination process, however.

Fungal contamination was found on the impression and smear preparations from the inner surfaces of the untreated incubator on all days >apart from the 7th and 35th day<.

In the case of the UV/ozone-treated incubator , contamination was to be observed on days 1, 7, 14 >but only prior to sterilization<. On all other days, no fungal contamination was to be found either before or after decontamination.

In the untreated incubator, fungal contamination was established in the 12 Petri dishes on every day throughout the test period. In each instance, 1 to 5 dishes were contaminated.

In the case of the UV/ozone-treated incubator, 1 to 3 Petri dishes were contaminated. On the 35th test day none of the 12 dishes were contaminated.

The fungal growth determined in both incubators >in Table 1< was again compared on the graph in Fig. 3.

The average germ level in the UV/ozone-treated incubator is only 1/5 of that in the untreated incubator.

Table 1, Fungal growth in the CO2 incubator

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