UDK 543.428.2:579.24	ISSN 1580-2949
Original scientific article/Izvirni znanstveni članek	MTAEC9, 44(3)153(2010)
DEGRADATION OF BACTERIA ESCHERICHIA COLI BY TREATMENT WITH Ar ION BEAM AND NEUTRAL OXYGEN ATOMS
UNIČEVANJE BAKTERIJ ESCHERICHIA COLI S CURKOM IONOV Ar IN NEVTRALNIH ATOMOV KISIKA
Kristina Eleršic1, Ita Junkar1, Aleš [pes1, Nina Hauptman2, Marta Klanj{ek-Gunde2, Alenka Vesel1*
1Jozef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia 2National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia alenka.vesel@ijs.si
Prejem rokopisa - received: 2009-06-18; sprejem za objavo - accepted for publication: 2010-03-12
Scanning electron microscopy was used to determine the difference between bacteria degradation by two types of particles presented in gaseous plasma, i.e. positively charged ions and neutral oxygen atoms. The source of ions was an argon ion gun with the ion energy of 1 keV and the flux of 3 X 1018 m-2 s-1. The source of neutral oxygen atoms was inductively coupled oxygen plasma supplying the flux of oxygen atoms of about 1.5 X 1023 m-2 s-1. The ion beam treatment time was 1800 s while the oxygen atom treatment time was 300 s. Bacteria Escherichia coli, strain ATCC 25922 were deposited onto well activated aluminum at the concentration of about 3 X 106 cfu and exposed to both particles. SEM analysis was performed using a field emission microscope with the energy of primary electrons of 1 keV. SEM images revealed huge difference in morphology of bacteria treated by both methods. While ions tend to drill holes into bacterial cell wall, the atoms caused a more even disruption of bacterial cell wall. The results were explained by kinetic, potential and charging effects.
Key words: bacteria, Escherichia coli, sterilization, degradation, oxygen plasma, atoms, ions, SEM
Z vrstično elektronsko mikroskopijo smo raziskovali razliko v degradaciji bakterij pri obdelavi z dvema različnima vrstama delcev v plinski plazmi: s pozitivno nabitimi in z nevtralnimi kisikovimi atomi. Vir ionov argona z energijo 1 keV in tokom 3 X 1018 m-2 s-1 je bila ionska puška. Vir nevtralnih atomov kisika s tokom 1,5 X 1023 m-2 s-1 na površino vzorcev pa je bila induktivno sklopljena kisikova plazma. Čas obdelave z ioni je bil 3000 s, medtem ko je bil čas obdelave s kisikovimi atomi 300 s. Bakterije Escherichia coli, sev ATCC 25922 smo nanesli na dobro aktivirano površino aluminija in jih potem izpostavili curkom obeh vrst delcev. Koncentracija bakterij je bila 3 X 106 cfu. Po obdelavi smo površino vzorcev analizirali z vrstično elektronsko mikroskopijo (SEM). SEM-slike so razkrile veliko razliko v morfologiji bakterij, obdelanih z atomi oziroma ioni. Medtem ko ioni povzročijo nastanek lukenj v celični steni bakterij, pa atomi bolj enakomerno degradacijo celične stene. Dobljene rezultate smo razložili z vplivom kinetičnih in potencialnih efektov ter vplivom nabijanja površine.
Ključne besede: bakterije, Escherichia coli, sterilizacija, degradacija, kisikova plazma, atomi, ioni, SEM
1 INTRODUCTION	type of gas or gas mixture, the pressure in the discharge
tube and the gas flow, the discharge power, the
Plasma sterilization has attracted much attention in	dimensions and the type of material used for the
the past decade due to possible application for steriliza-	discharge chamber, etc. Much less work, however, has
tion of delicate materials that cannot stand autoclaving in	been done on determination of sterilization effects versus
humid air at 130 °C. Several different types of discharges	plasma parameters. Not surprisingly, the explanations of
have been used to create plasma suitable for destruction	observed sterilization effects are often contradictory.
of vital bacteria and their spores.1 9 The discharges in-	Many authors explain sterilization by destruction of
clude low and atmospheric pressure. Among atmospheric	bacterial DNA caused by UV photons from plasma.
discharges, RF and microwave plasma torches are partic-	Other authors state that sterilization is due to chemical
ularly popular, while the dielectric barrier glow dis-	etching of the bacterial cell wall with radicals such as O,
charge was not found as efficient. The same applies also	N, H, etc. Some other authors take into account also the
for otherwise popular corona discharges. The low pres-	kinetic effects of bombardment with positive ions, and
sure discharges suitable for destruction of bacteria at low	most authors agree that synergetic effects play an
temperature include the DC, RF and microwave dis-	important role.
charges.10-14 Radiofrequency discharges are particularly	In order to understand the role of different plasma
popular since they assure for a high density of plasma	particles it is the best to separate them and treat bacteria
radicals and rather low kinetic temperature of neutral	only with one type plasma particles. At the experiments
gas.	presented in this paper we exposed bacteria separately to
Most authors presented results on bacterial	2 types of different plasma particles: energetic non-reac-
deactivation as a function of discharge parameters. The	tive ions and neutral oxygen atoms with the kinetic tem-
discharge parameters that are often varied include the	perature of 300 K.
2 EXPERIMENTAL
2.1	Sample preparation
Bacteria Escherichia coli (E. coli) were cultivated according to the standard procedure. In experiment we used bacteria E. coli strain ATCC 25922. It was grown at 37 °C, on LB plates for 24 h. Cells were then resuspend-ed in sterile water. Number of cells was adjusted to approximately 3 X 106 cfu (colony forming unites).
Live bacteria were deposited onto commercially available aluminum foils. Substrates were first carefully cleaned with wet chemical treatment, and then activated with a brief exposure to oxygen plasma in order to assure the removal of any traces of organic contaminants and achieve optimal hydrophilicity. A drop of water containing vital bacteria was placed onto the substrate. Due to highly activated surface, the bacteria-containing water drop was spread on a large surface. Such spreading allowed for two dimensional distributions of bacteria with out overlapping.
2.2	Experimental system
Samples were treated either by neutral oxygen atoms in an afterglow chamber of oxygen plasma reactor or by positively charged Ar ions from a commercial ion gun. The schematic of the experimental setup for the case of oxygen atoms is shown in Figure 1. The vacuum system is pumped with a two stage rotary pump. The effective pumping speed at the exit of the experimental chamber is almost identical to the nominal pumping speed of the pump, i.e.16 m3/h. The experimental chamber is connected to a discharge chamber through a narrow tube that allows for a difference in the effective pumping speeds between the experimental and discharge chambers and thus a pretty high drift velocity of gas through the narrow tube. Both chambers as well as the connection tube are made from borosilicate glass Schott 8250. This glass has a low recombination coefficient for the reaction O + O ^ O2.15'16 Such a configuration assures for experiments at constant (i.e. room) tempera-
Figure 1: The experimental setup for treatment of bacteria with neutral oxygen atoms. 1 - vacuum pump, 2 - experimental chamber, 3 - discharge chamber, 4 - sample, 5 - vacuum gauge, 6 - catalytic probe, 7 - inlet valve, 8 - oxygen flask
Slika 1: Shema eksperimentalnega sistema za obdelavo bakterij z nevtralnimi atomi kisika: 1 - vakuumska črpalka, 2 - eksperimentalna komora, 3 - razelektritvena komora, 4 - vzorec, 5 - vakuummeter, 6 -katalitična sonda, 7 - dozirni ventil, 8 - jeklenka s kisikom
Figure 2: The experimental setup for treatment of bacteria with Ar ions: 1 - UHV chamber, 2 - pumping system, 3 - vacuum gauge, 4 -sample, 5 - ion gun, 6 - energetic ions.
Slika 2: Shema eksperimentalnega sistema za obdelavo bakterij z ioni Ar: 1 - UVV komora, 2 - črpalni sistem, 3 - vakuummeter, 4 -vzorec, 5 - ionska puška, 6 - energijski ioni
ture and constant density of oxygen atoms in the vicinity of substrates. The density of neutral oxygen atoms is measured with a catalytic probe.17-19 At the experimental pressure of 75 Pa the O density is about 1 x 1021 m-3. The resultant flux of neutral oxygen atoms onto the surface of the sample is then j = ^ nv = 1.5 x 1023 m-2 s-1.
The experimental setup for treatment of bacteria with Ar ions is shown schematically in Figure 2. The source of Ar ions is a commercial ion gun used for sputtering of materials during depth profiling. Ar ion beam with the energy of 1 keV at an incidence angle of 45° and a raster of 3 mm X 3 mm was used for treating bacteria. The ion current is 0.15 A/m2 giving the ion flux onto the surface of the substrate with bacteria of 3 x 1018 m-2 s-1. We used no charge compensation during treatment of bacteria with argon ions.
2.3 SEM imaging
Scanning electron micrographs of substrates with bacteria were obtained using a field emission microscope Karl Zeiss Supra 35 VP. A 1 kV accelerating voltage was used to record images.
3 RESULTS
SEM image of untreated E. coli bacteria is shown in Figure 3. The image does not look very sharp. This is not an artifact of the microscope but rather the consequence of the presence of the capsule on the surface of bacteria as well as between bacteria. Namely, the capsule is composed predominantly of chemically bonded water as well as some sugars, proteins and lipids - material that are a bad scatterer for electrons. That's why the SEM image looks rather dim.
A SEM image of a bacteria treated by Ar ions is shown in Figure 4. The bacteria are badly damaged and definitely not capable of revitalization.
Figure 3: SEM image of untreated bacteria Slika 3: SEM-slika neobdelane bakterije
A SEM image of bacteria treated in the afterglow of the oxygen plasma, i.e. with neutral oxygen atoms only, is presented in Figure 5. In this case, the surface morphology is very different from that observed in Figure 4.
4 DISCUSSION
Figures 3, 4 and 5 represent SEM images of bacteria E. coli. Bacteria presented in Figure 3 are live what has been confirmed by cultivation using the standard plate count technique. Bacteria are covered with a thin film of jelly of lipopolysaccharides and is called capsule. The majority of lipopolysaccharide cover material has chemically bonded water. This thin cover is (about 400 nm or more) capsular polysaccharide gel20 which serves as a medium for gluing bacteria together as well as for sticking onto surfaces. The capsule also facilitates formation of three dimensional clusters of bacteria. Such clustering was not observed at our experiments since we activated the surface of the aluminum prior to bacterial deposition. The surface of activated aluminum foil is perfectly hydrophilic thus allowing for two- dimensional spreading of bacteria on its surface. Such procedure for bacteria fixation therefore allows for uniform treatment of bacteria with plasma particles.
An exposure of bacteria to argon ions causes a strong damage. Figure 4 represents the SEM image of bacteria
Figure 4: SEM image of bacteria treated with argon ions Slika 4: SEM-slika bakterije, obdelane z ioni argona
Figure 5: SEM image of bacteria treated with oxygen atoms Slika 5: SEM-slika bakterije, obdelane z atomi kisika
after receiving the argon ion dose of 5.4 x 1021 m-2. The bacteria are definitely not capable of revitalization what was proved also by control experiments using the plate count technique. It is interesting that the damage caused by ions is far from being uniform. Namely, a hole - like structure of the bacterial cells is observed. Although it is known that ion beam etching is never perfectly homogeneous and isotropic, such rich surface morphology cannot be due to common effects observed at ion beam etching of organic materials. The observed morphology may be attributed to appearance of the local surface electrical charge during treatment with positively charged ions. Namely, the electrical conductivity of bacteria is poor. Since the composition of the cell wall is far from being uniform, some spots on the surface may keep larger charge than other. The surface charge influence the local uniformity of the ion flux on the surface causing local focusing and thus further non-uniformity of the ion beam etching. Finally, the bacteria obtain morphology as shown in Figure 4. The ions practically cannot reach the uppermost part of bacteria since positive charge prevents it.
The SEM image of bacteria treated with oxygen atoms shows a completely different picture. In this case, the badly damaged bacteria are flattened, also. In fact, little material remained after receiving the dose of approximately 4.5 x 1025 m-3. The remains observed on the surface of the aluminum foil after treatment with oxygen atoms represent only ash - mostly inorganic remains of the bacterial material after rather complete oxidation of organic material. This picture is in agreement with previous observations on selective etching of organic materials by oxygen radicals21.
5 CONCLUSIONS
Bacteria E. coli were deposited onto aluminum foils and exposed to positively charged argon ions or neutral oxygen atoms in the ground state. In both cases, the samples were kept at room temperature. Since argon is inert gas that does not interact chemically with organic material, the interaction was almost completely kinetic. Apart
from radiation damage, the argon ions caused sputtering of the bacterial material. The sputtering was extremely inhomogeneous what was explained by local charging of the bacteria. In the case of oxygen atoms, any kinetic effect is neglected since the O atoms are thermal at room temperature. In this case, rather uniform degradation of bacteria occurred and only ashes remained after the treatment. The interaction of O atoms with bacteria is therefore purely chemical. In both cases, bacteria were badly damaged and unable to revitalize.
ACKNOWLEDGEMENT
This research was funded by Slovenian Research Agency, Contract No. P2 - 0082.
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