Risks and benefits assessment of UV light system in Biosafety Cabinet

By Dian Susanti

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Introduction

From the end of 1980, when the sunlight spectral bands has successfully mapped, the following research has

brought the proof that the ultraviolet as sunlight component identified as biocide agent. This chronology leads to

UV bulb creation and development, germicidal research and up to advanced microbial control system it is by

employing UV energy in certain hygiene area. Nowadays, UV radiation is widely utilized in water sterilization to

control bacterial growth, clean air technology to create microbial-free area, healthcare equipment and facility

disinfection in infection control.

It has been years since UV light applied in Biosafety cabinets (BSC) as surface disinfection agent, this feature is

not an absolute requirement in BSC system. UV setting has been guided by NSF/ANSI – 49 in chapter 5.25.2

and G.3.3.3 stated that UV lighting is not recommended to be installed in Class II BSC, because the usefulness

subject for debate among many users and manufacturers. If necessary, it shall be installed in such manner that

is does not reduce required performance.

Many BSC users are insist to equipped their cabinet with UV system and manufacturers have to be aware if

their BSC properties could be experiencing significant effect due to major airflow disturbance issue. As

manufacturer, Esco has to accommodate our valued customers wish, it is by doing risk and benefit assessment

of BSC-UV integration system, so the both sides appeal will be retain in some melting jar, and the best choice

would be obtained. Esco UV lamp emitting 253.7 nm, this wavelength is including in UVC wavelength range, so

the following experiment and discussion will be more concern about this matter.

Esco UV light properties

UV light is conventionally generated in mercury vapor discharge lamps. There are few types of discharge lamps

all with different characteristics. Low pressure lamps emitting UV energy at 253.7nm and the medium pressure

lamp produces a continuum of energy in the bactericidal region 200 to 315nm.

Esco Biosafety cabinet is equipped with germicidal UV lamp emitting UV rays at 253.7 nm, and this wavelength

is classified as UVC. The shortwave UVC rays are absorbed in certain area of the hereditary substance in the

cell nucleus (DNA) and as a consequence photo-chemical changes occur DNA bases become unusable for

copying processes by itself. If the number of destroyed carrier information to a particular mass is high enough,

and then the cell line will die without having duplicated. Due to the high level of UV output and its lack of

sensitivity to water temperature, medium pressure lamps are usually utilized in all but the smaller flow

applications. The UV output of a low-pressure lamp is sensitive to the temperature of the lamp, which in turn is

dictated by the temperature of the surface being treated. For this reason, care must be exercised when

specifying a UV unit to treat biosafety cabinet surface at UV manufacturer recommendation.

For Esco BSC, UV lamp’s glass is made of so called soft glass material. This special glass envelope filters out

ozone-forming radiation, in this case the 185 nm mercury line. There is no ozone forming radiation can escape

from the bulb, so there is no ozone release to the work surface, which most likely will cause a significant level of

material deterioration placed inside of BSC chamber. Therefore, the contaminants will just killed by UV energy,

not by ozone saturation inside of work zone chamber.

UV Limit Value and Effect to Human and Materials

UV germicidal lamp irradiation is useful only for surfaces sterilization and some transparent objects. Many

objects that are transparent to visible light are absorbing UV rays. UV irradiation is routinely used to sterilize the

interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under dirt

(which may become polymerized after prolonged irradiation, so that it is very difficult to remove). It also

damages some plastics, such as polystyrene foam if exposed for prolonged periods of time.

UVC rays are the highest energy of all UV rays and potentially could be the most harmful to eyes and skin.

Some fixture should be made to block this shortwave UV. For Esco this fixture is built with interlock switches

that prevent operation with the lamp exposed. When biosafety cabinet is on operation, it is set that UV lamp will

deactivate by itself after UV decontamination mode. Moreover, even the operator is mistaken to press UV to be

ON, the lamp won’t shine out. Ordinary glass as BSC sash window/glass does stop this dangerous UV, as well

as all other UVC and UVB wavelengths.

Sash Glass Properties (Esco UV system, killing inside, protecting to the outside)

Esco’s BSC is equipped with tempered laminated glass with different thickness and models for various type of

BSC. Compared to common glass sheet, laminated glass sheet is stronger and can withstand more impact.

Even the laminated glass broken, the fragments will keep sticking on the interlayer and won’t scatter. This glass,

has UV filtering ability, the interlayer can filter out ultraviolet rays, so that the UVC energy won’t penetrate out of

the BSC. Then, UV ray emission is minimized as lower as possible to the outside preventing furniture from

fading and excessive exposure to human body.

Materials and methods

Microorganism. There are 6 types of bacteria and 3 types of yeast were used in this experiment.

Microorganisms used were all came from Presque Isle Culture collection. The bacteria cultures used are

Staphylococcus epidermidis; Serratia marcescens; Enterococcus faecalis; Escherichia coli, while the yeast were

Saccharomyces cerevisiae, Candida albicans and Rhodotorula rubra. Each of bacteria and yeast were

refrigerated at 4°C in Tryptone soya and saboraud agar slant.

In vegetative cell preparation, bacteria cultures were extracted from their agar plate, and grown aerobically in

tryptone soya broth. Yeast cultures in agar slant were also transferred into saboraud dextrose broth and grown

aerobically for72 hour at 25°C. All cultures were harvested at their stationary growth phase. Vegetative cells

were purified by extracting the cell from growth media via centrifugation process. The harvested cells were

resuspended in sterile phosphate buffer saline at pH 7.3 and stored in 4°C prior UV exposure procedure.

Bacillus subtilis var globigii and Geobacillus stearothermophilus spore were obtained as ready-to-use spore

suspension. Prior UV treatment spore suspension was diluted to desired concentration in sterile phosphate

buffer saline at pH 7.3. Initial concentration of B.subtilis var globigii and G.stearothermophilus is 1.5 x 1010

and

1.3 x 108.

Biosafety Cabinet Setting. In this experiment Esco Labcuture® Plus was used, factory standard UV bulb was

attached to the cabinet accordingly, and cleaned up prior the test. The cabinet was turned ON and sash window

was pulled up to defined height, then warmed up for 3 minutes. Insert UVX Radiometer into the cabinet, put

down the sensor on the work zone tray, and then set the sensor to zero. Later, sash window was pulled back

down, and the UV button was pressed to set the UV bulb to glow. UV intensity was measured by leaving the UV

sensor to record the intensity value for 1 minute of each site as sketched in figure 1.

Figure 1. UV Sensor Location for UV Intensity Mapping

UV Decontamination Procedure. To conduct a quantitative experiment, number organisms exposed to UV

have to be sampled in the same amount with applied microorganisms prior UV exposure, this way we could

retrieved the representative result. Therefore, a sterile stainless disc was used as container of tested

microorganism. The disc was put in the work zone tray divided by rear, center and front area, as shown in figure

2. Disc placement was stated as location 4 of each rear, center and front area. Disc locations are shown clearer

in figure 3 below. Cell suspension was put in each disc, and then UV was set to glow for 60 minutes. The

elapsed time used was and then cell recovery could be measured. Dilution samples were spread into tryptone

soya agar and incubated for 48 hr at 25°C.

Figure 2. Disc location exposed to UV Light

Result and Discussion

UV light is preferred method for sterilization in different fields especially in laboratories, dental medicine and

cosmetics, due to destroying effect and different efficiencies of disinfectant on materials. Time and surface

properties appear to be important in UV sterilization. In addition to that, wavelength of UV light and distance

between the lamp and material are associated with bactericidal, fungicidal and sporocidal effect of the UV light.

The distance issue represent to the intensity of UV energy exposing the contamination agent to achieve

expected sterility level of Biosafety cabinet.

UV intensity measured at the work zone area was read as values which are significantly different from each site

as displayed in the below Figure3. After that, the challenged microorganisms were put in the center area of the

work tray, and sterilized by UV rays with result as shown in table 1. From this experiment known that log

reduction of each microorganism is different

This result indicating that kill achieved in every site is not uniform. In killing experiment, we put challenged

microorganisms in the center of the work tray, as representative of the highest killing result of each tested row.

From figure 3, shown that distance differences of each disc from UV source.

.

Figure 3. UV Light Intensity Mapping in The Workzone Tray

Table 1. UV Decontamination Result

Microorganisms Initial concentration Log reduction

(cfu/ml) Front Center Rear

Bacteria vegetative cell Staphylococcus epidermidis 2.0 x 10

5 4.60 5.00 5.30

Enterococcus faecalis 2.3 x 105 4.66 5.20 5.36

Escherichia coli 1.3 x 106 5.11 5.81 6.10

Serratia marcescens 3.0 x 106 5.47 6.10 6.48

Bacteria spores Bacillus subtilis var.globigii 5.0 x 10

4 2.00 2.30 2.60

Geobacillus stearothermophilus 1.8 x 104 1.65 1.65 2.25

Yeast vegetative cell Sacccharomyces cerevisiae 5.5 x10

4 4.74 4.74 4.74

Candida albicans 1.7 x 104 4.23 4.23 4.23

Rhodotula rubra 1.6 x 104 4.20 4.20 4.20

Table 1 show that bactericidal effect was determined after 60 minutes for S.epiderimidis, E.faecalis, E.coli, and

S.marcescens for all site of exposure. For C.albicans, S.cerevisiae and R.rubra, fungicidal effect was also seen

after 60 minutes. Complete sterilization applied for all bacterial and yeast vegetative cells with disc located in the

rear area of the work zone with 230 mm of distance from UV bulb.

Front and rear location of discs were experienced bacteristatical and fungustatical effect, vegetative cells on

these disc most likely were inactivate or having incomplete killing, which later resulting some recovered

colonies. UV intensity those exposing the front and center area of the work zone were significantly lower than

the rear area. We could say that UV intensity ratio of these three locations is 1:2:3, this is not same with

decontamination result from each location. Qualls and Johnson (1983) reported that there were several

problems with UV methods dose estimation used for disinfection of water. Background microbial population in

liquids along with particles and organic matter has been associated with low transmission of UV light. Hence,

contamination risk cannot be disregarded in case of insufficient UV sterilization, which is associated with

exposure of light to material. In this study, lower-class fungal, bacterial and spore suspensions were used,

separated from growth media to make sure the highest transmission was applied in this procedure, to assess

UV light application in different intensity of representative area.

UV energy required to kill representative microorganisms is vary among literatures, and most likely the data was

resulted from experiment conditions differences. Growth nutrient and special compound in growth media

microorganisms would affect cells phenotype, and then resulting in different UV absorbtivity value. Enclosure

humidity is also contributing in this killing effect. UV rays are having poor ability to penetrate into the deep water,

environment that provide high moisture will not achieve good killing in UV decontamination.

In this experiment, cell’s macroscopic observation was performed after microbial suspension was exposed the

UV ray, noticed that the suspension dried as looked like that UV rays were transmitting some heat energy to the

disc. Water as cells media and water as cell composition were sucked up and observed as sun rays effect to the

disc, and log reduction came from cells damage. As human body, microorganisms cells is also contain a lot of

water and contributing to different cells form of each microorganisms, based on the water arrangement in cell

composing process. UV ray may cause severe burnt to human body and eyes,

Figure 4. Test Microorganisms Placement

Many clinical isolate of Candida albicans displayed strongly biased auxothroph spectrum after ultraviolet

irradiation. This was interpreted as the consequence of the natural heterozygosity of many C.albicans strains for

some loci. This hypothesis was later confirmed from the analysis of sectored colonies obtains after UV treatment

of putative natural heterozygotes or revertants from mutants isolated by chemical mutagenesis. This later

resulting gene alteration in some loci which would affect phenotype of C.albicans cells such as growth disorder

as result of DNA base changing in some in protein framework.

In this experiment, mutation effect prominently observed from S.marcescens colony, recovered cell grown in the

agar was changing color into yellowish white from previously cultured as in red to pink color. In this case, gene

expression for controlling pink colorization of colony appearance was shut down by UV light. No special

changing macroscopically observed from the rest of E.faecalis, S.epidermidis, E.coli, spore derived culture and

all of the yeast species colonies.

Serratia marcescens

Serratia marecescens after UV radiation

We are, as the one manufacturer that providing UV system applied in our BCS, does not pull over from

advantages and disadvantages of UV usage. As previously stated in the above, UVC will be harmful if directly

exposed to human body or furniture material. At certain dose this action may lead to keratoconjunctivis, or

corneal burn and skin cancer. We are controlling this potential risk by providing protective sash window to our

cabinet and utilizing interlock system to operate the UV to glow. Because of UV bulb attached to the rear wall of

Biosafety cabinet, this may disrupt airflow system preferably protection at the back side of workzone, which is in

line with UV setting. User may take off the UV bulb when operating BSC at normal mode, but this action is not

feasible for users as the UV bulb is made from glass material. Accident may happen when the bulb is not

properly stored and causing danger to users. Talking about efficiency, certain species or condition of many

contaminants preferably may have resistance property to UV light, they are organisms that having photo

reactivation system in their cells, or in survival ability when they are being treated with UV light such us cyst and

spore form.

Utilizing UV system in BSC has some major advantages for disinfection application. First, unlike other

disinfectant UV application leaves no residue, as physical disinfectant action begin upon energizing the bulb.

Ultraviolet light is an effective germicide and virucide for organisms directly exposed to UV light. As stated in the

previous session, the UV inactivation doses have been determined for variety of organisms. UV is more efficient

for vegetative organisms and viruses. By using NIH/CDC criterion of minimum acceptable irradiance in a

biosafety cabinet of 40µW/cm2, it takes 12.5 minutes to reach the 30,000 µJ/cm2 found to inactivate spore

forming organisms. Use of a UV light in excess of an hour or overnight is massive overkill. Moreover, each UV

system has its own shelf life, as UV system utilized in Esco BSC is having average life of 9000 hours. More than

this, users have to ask for replacement for UV bulb used.

The disinfection between UV energy & population reduction

The disinfection is based on of UV energy applied, this is usually referred to as dose and it is calculated as

intensity times the application time. The units of UV dose usually mentioned as µJ/cm2, it is equal with 1000

µW/cm2. In addition the effect o UV dose on the organism is dependent on the species it self. As simple

bacteria like Eschericia coli require relatively small doses of energy and other microorganisms such Hepatitis A

virus can require higher levels. Raising or lowering the dose for specific organism has a logarithmic effect such

that doubling the dose for a 90% kill will give a 99% kill. The survival rate for a given organisms is therefore a

function of the initial numbers of organisms and the applied dose.

Conclusion

UV usage in Esco biosafety cabinet will be achieved by: sash glass with UV filtering ability, interlock switch

system of UV operation, and after and between uses UV program activation. In UV sterilization of BSC

enclosure humidity of air and UV intensity is largely contributing to effective sterilization result.

References

Kowalski. Wladyslaw. 2009. Ultraviolet Germicidal Irradiation Handbook; UVGI for Air and Surface Disinfection.

Springer-Verlag. Berlin Heidelberg.

Halfmann.H., Denis.B., Bibinov. N., Wunderlich. J. Awakowicz.P. 2007. Identification of the most efficient

VUV/UV radiation for plasma based inactivation of Bacillus atrophaeus spores. Journal of Physics D: Applied

Physics.

Reynolds. A. Kelly. 2002. Ultraviolet Light; An Alternative Disinfectant. Water conditioning and purification

magazine international Volume 44, No: 6.

Ozcelik. Berrin. 2007. Fungi Bactericidal and Static Effect of Ultraviolet Light in 254 and 354 nm wavelengths.

Research Journal of Microbiology, Volume 2, No 1, Page 42-49.

A primer UV-C light. http://hygienitech.com/Hygienitech%20UV-C%20Light%20Primer.pdf

Moat. G. Albert, Foster. W. John. 1995. Microbial Physiology; Third Edition. Wiley-Liss Publication. New York.

Molero. Gloria et. al. 1998. Cadida albicans: genetics, dimorphism and pathogenicity. International Microbiology

volume 1, page 95-106

Qualls. R.G and Johnson. J.D. 1983. Bioassay and Dose Measurement in UV Disinfection. Applied

Environmental Microbiology, p 872-877.

Riesenman. P.J and Nicholson. W.L. 2000. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation. Applied Environmental Microbilogy p 620 – 626.