FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
As amended by Patents (Amendment) Apt, 2006
&
the Patents Rules, 2006
PROVISIONAL / COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
"Wall and floor mountable, Portable, Rapid, Air Based Sterilizer For The Inhibition of Superbug, Suitable In Medical Applications"

[001 ] FIELD OF THE INVENTION
[002] The present invention is related to the disinfection/inhibition of airborne bacteria using reactive species generated from atmospheric pressure miniaturized dry sterilizer. This device can be mounted in the form of floor tile or wall mount, which is most suitable for the inhibition of growth of superbugs present in Intensive Care Units (ICU) of hospitals/medical care unit or laboratories.
[003] BACKGROUND OF THE INVENTION
[004] Nosocomial infections (hospital acquired infections) and microbial aerosol are transmitted through various vectors such as through sneezing, coughing, respiration, insertion of medical devices, interaction with different persons such as hand shaking or during the change in the weather conditions. These aerosol/superbugs can easily migrate along the direction of air flow. In 2020-21 world has faced dangerous effects due to spread of corona virus. Even the superbugs stocked to the footwears as well as other medical wear such as mask, gloves, apron etc. easily migrate from one place to other. The demand of such medical wear increases dramatically, when the spread of infectious disease is enormous.
[005] Since many years it is one of the major causes of infection arising from survival of bacteria/virus/fungi grown onto the surface of living organism or non-living objects. The superbugs generated in Intensive Care Units (ICU) or operation theaters are easily transmitted and those are difficult to be controlled. Scientists and doctors are in search of suitable alternatives to inhibit the bacterial growth and their transmission to the human body.
[006] Therefore, novel methodologies, which are non-toxic or less toxic are desperately needed. Non-antibiotic approach for the prevention and protection against the infectious diseases are highly encouraged. To explore these possibilities, new approach such as development of dry disinfectant devices for therapeutic treatment is one of the upcoming research area.

[007] Felix Theinkom reported antibacterial efficacy of dry reactive species based device against the growth of Enterococcus faecalis planktonic cultures1.
[008] Tendency of certain bacteria to adhere on the surface and subsequent growth to form biofilm is one of the major limitations on the utility of the dentistry implants. Myung-Jin Lee et al reported use of dry reactive species for the treatment of titanium surface suitable to inhibit the growth of dentistry bacteria .
[009] Vladimir Scholtz et al reported in a review article, various methods suitable for the generation of dry reactive species and their utility for bacterial, fungal, viral and biomolecule inactivation have been discussed .
[010] Marlies Govaert reported that efficacy of dry reactive species generator for the inhibition of biofilms. They configured devices and used it for disinfection of L. monocytogenes and S. Typhimurium biofilms .
[011] Mingming Huang et al reported use of dry disinfectant device to investigate cellular damage in Salmonella Typhimurium and Staphylococcus aureus. It was noted that the device is more effective for Gram negative bacteria than Gram positive bacteria5.
[012] Alexander Wright et al used micro-bubble dry reactive species generator to produce reactive oxygen species as well as reactive nitrogen species suitable for disinfection of E. coli mixed with effluent.
[013] Pradeep Puligundla et al used dry reactive species generator for the disinfection of food borne pathogens namely glass, polyethylene, polypropylene, nylon, and paper foil and different types of packaging materials. The device was operated at 10 kV, 208 W and used for the deactivation of E coli, Salmonella typhimurium and Staphylococcus aureus7.
[014] Sang Hye Ji used dry reactive species generator for the disinfection of Escherichia coli and Staphylococcus aureus on contaminated perilla leaves by cylinder type device with underwater bubbler treatment. The emission

spectra indicate presence of N2 in near-UV region (300-400 nm) along-
with weak signature of NO species8. [015] Fatemeh Rezae et al reported use of dry reactive species generated for the
surface modification of polymethyl methacrylate (PMMA) and further
used to improve its biocompatibility and antibacterial activity . [016] Haiping Liu et al used graphitic carbon nitride nanosheet for efficient
photocatalytic pathogenic bacteria disinfection . [017] Miri Park et al used microorganism-ionizing respirators with reduced
breathing resistance to remove airborne bacteria. The ionizing respirator
mainly produces ozone which was used for the disinfection of air born
bacteria. Finally manganese oxide mesh was used to reduce the ozone
concentration after the use for disinfection . [018] In view of developments in the area of nanotechnology, the nano particles
are also helpful to the decontamination of microorganisms. Dawei et al
reported the effect of iron oxide nano-wire based filters for inactivation of
12 airborne bacteria , also silver doped titanium dioxide as a photocatalyst
1 3 used for indoor bacteria control . Inactivation of S. Epidermidis, B.
subtilis and E. Coli deposited on to a filter using the silver nano-particles.
In their study the bio-aerosol deposited on to the filter were exposed to the
airborne silver nanoparticles and aerosol measurement devices were used
to understand physical and biological properties, which indicates their
utility as a bio-aerosol control methodology . Inhibition of gram positive
and gram negative bacteria using Si-Si02 nanostructure has been reported
by our group .
[019] It has been recently reported that dry reactive species generator, which are
operated under normal temperature and pressure conditions, can replace
the traditional sterilizer. Michael et al reported the air based dry reactive
species generator device and showed that the device generates active
species like ozone, UV radiations, OH radicles and atomic oxygen actively
participate to inhibit the bacteria. Exposure to ozone leads to a 3.5 log (i.e.
99.95%) reduction in the cultured bacterial colony . The operating

condition reported mimics the situation that might exist during the bio-terrorist attack like release of high concentration of bio-aerosol with a high flow rate. Author emphasizes the need of advance sterilizer, which can demonstrate the sterilizing effect on microorganisms16.
[020] A co-planer dry reactive species generator device has been reported by Evs et al. It mentions that the stress developed on E. Coli bacteria in the humid air after the plasma exposure affect the metabolic arrest and helps to inactivate the bacterial growth .
[021] E Timmermann and Miri Park have designed ionizing respirator
respectively which has been shown to be useful in removing the airborne
11,18 bacteria and reduces the breathing resistance ' .
[022] Based on the guidelines for the DSHF (Disinfection and Sterilization in Healthcare Facilities, 2008),
[https://www.cdc.gov/infectioncontrol/guidelines/disinfection/] the use of oxygen reactive species, nitrogen reactive species, other reactive species as an effective on MRSA (superbugs)19 in the vicinity of the device, but the effect is inadequate for the hospital side room.
[023] OBJECTIVES OF THE PATENT
[024] The present invention comprises of all the objectives as listed herein under, of which
[025] It is preliminary objective of the present invention, to develop a device that can be fixed in place of tile at the entrance of building/room or wall mount or any other suitable mount of individual ward/ICU/operation theater of the hospital, medical company, pharma food and beverages, clean rooms and storage spaces etc.
[026] It is another objective of the present invention in addition to aforesaid objective(s) that the reactive species generator device is suitable to inactivate the bacteria adhered onto the used mouth mask by hospital medical staff/ sweeper staff/ front line staff or other staff.
[027] It is another objective of the present invention in addition to aforesaid objective(s) to elucidate the method to produce reactive species without

using complicated and bulky components, vacuum pumps, reactor or other similar components further to ease the handling of prototype.
[028] It is another objective of the present invention in addition to aforesaid objective(s) that the method produces sufficient quantity of reactive oxygen, nitrogen and other species within few tens of seconds.
[029] It is another objective of the present invention in addition to aforesaid objective(s) that the method so provides reactive species suitable for inhibition of the growth of bacteria viz E coli, Staphylococcus and others.
[030] It is another objective of the present invention in addition to aforesaid objective(s) that the method produces ozone as an example of reactive species, which sanitize the surface and inhibit growth of many microbial and viral infections.
[031] It is another objective of the present invention in addition to aforesaid objective(s) that the device developed is small, portable, and cost effective as it does not require expensive consumables except electricity.
[032] These and other objectives of the present invention will be lucent to the reader upon detailed disclosure.
[033] DETAILED DESCRIPTION OF THE INVENTION
[034] The present invention is explained herein under with reference to following drawings, in which,
[035] Figure 1 shows the schematic of isomeric view of cascaded reactive species generator unit. The basic unit is as discussed in Figure 4-6. The same unit is connected in cascaded manner to enhance the concentration of reactive species, which can be mounted on wall, floor, or any other suitable place.
[036] Figure 2 shows the schematic of top view of cascaded reactive species generator unit as shown in Figure 1.
[037] Figure 3 is a schematic top isometric view of the single unit out of the cascaded unit shown in Figure 1 and Figure 2, fixed in a box along with other required components and used to generate required dry reactive species according to an embodiment of the present invention.

[038] Figure 4 is a isomeric view of the single unit out of the cascaded units shown in Figure 1 and Figure 2, which is actually used to generate dry reactive species according to an embodiment of the present invention.
[039] Figure 5 is a schematic left cross sectional view of the dry reactive species generator unit according to an embodiment of the present invention.
[040] Figure 6 is a schematic top view of the dry reactive species generator unit shown in Figure 4, according to an embodiment of the present invention.
[042] Figure 7 is concentration of ozone generated at the place where the sample to be disinfected is kept, as a function of device TURN ON time (sec) recorded using ozone meter.
[043] Figure 8 is photograph of E coli grown in agar medium having initial concentration with 10" dilution (with bacterial culture of 0.18 Optical Density (OD)) with and without treatment by reactive species generated using the device as described in Figure 3-6, for the duration of a) 0 sec, b) 60 sec. c) 120 sec. d) 300 sec, e) 600 sec and f) 900 sec. The photographs were recorded after 24 h of incubation time at 37 °C. Two set of results are shown.
[044] Figure 9 is photograph of Staphylococcus aureus grown in agar medium having initial concentration with 10" dilution (with bacterial culture of 0.18 OD) with and without treatment by reactive species using the device as described in Figure 3-6, for the duration of a) 0 sec, b) 60 sec. c) 120 sec. d) 300 sec, and e) 600 sec. The photographs were recorded after 24 h of incubation time at 37 °C. Two set of results are shown.
[045] Figure 10 shows photograph of mixed bacteria collected from foot ware, grown in agar medium having initial concentration with 10" dilution without and with treatment by reactive species using the device as described in Figure 5-6, for the duration of a) 0 sec, b) 30 sec. c) 60 sec. d) 120 sec. The photographs were recorded after 24 h. of incubation time at 37 °C. Two sets of results are shown.
[046] In the drawings included here the same references and symbols have been used throughout in order to refer to a particular part wherever possible.

References given in particular example are illustrative only, and do limit the scope of the invention or the claims. For the clarity purposes, not every component is labeled in every figure, nor is every component an embodiment of the invention shown. [047] Detailed description
[048] The present inventors propose herein is based on the use of air-based
sterilizer (dry sanitizer) which is small, portable, and cost-effective device.
Here the device works at normal temperature and pressure conditions. The
temperature of the device as well as the region of reactive species
possesses the temperature in the vicinity of room temperature. The device
not only generates oxygen plasma species but it also produces other
reactive species of nitrogen such as N+, NH etc. It also emits UV A and
UV-B radiations.
[049] The device enclosed in box has provision to subject the micro-organism in
direct or indirect contact with the reactive species. In direct contact the
microorganisms are exposed to air based reactive species including oxygen
and nitrogen-oxygen species along with UV-A and UV-B radiations,
whereas in indirect contact the microorganisms are exposed to mainly
ozone species. The typical time of reactive species interaction varies in the
range of few second to few tens of seconds, which inhibits growth of
microorganism up to 60 to more than 90 %. Even after the treatment on the
microorganism the temperature of the device remains in the vicinity of
room temperature. The device does not require any cooling arrangement,
natural cooling is sufficient. The device consumes power of few Watt/min
for its operation where the signal applied is pulsed. The device generates
reactive oxygen species as well as reactive nitrogen species.
[050] The unit device can be cascaded in two or three dimensions in order to
increase the concentration of reactive species and obtain desired
disinfection. Such cascaded device can be fixed as floor tile or wall mount.
Attention of the interested person is requested to the detailed description of

the present invention which narrates all the details of the device development.
[051] The advantages of current prototype are: a) it can generate reactive species in a few second, b) the device is portable, c) it produces dry reactive species, d) it has low cost, e) it consumes low power, and f) requires low maintenance cost.
[052] The prototype consists of acrylic sheets, conducting mesh, and ignition transformer.
[053] Micro-ionization of air molecules occur at atmospheric pressure based on the mechanism associated with the Paschen curve. A micro discharge follows the Lissajous curve . The dissociation, ionization and recombination process are operative till the power is applied across it. Different species like electrons, ions, OH radicals, atomic oxygen and ozone etc. are generated. UV radiations are also generated in this process. The reactive species are responsible in the present disinfection study.
[054] Example 1: Miniaturized air-based tile sterilizer
[055] For the purpose of disinfection of the superbugs in a working space; a floor and wall mount that actively sanitizes the room through ozone or other similar action has been described. Ozone works as reactive species to sanitize the surface of any microbial and viral infections. The miniaturized air-based sterilizer is cascaded so as to increase the concentration of reactive species. The methodology used in the present invention does not require any extra consumable except bare minimum electricity. Figure 1 and Figure 2 show the isometric and top view of cascaded air based sterilizer unit. [056] Such cascaded unit can be used in medical industry, pharma, food and beverages, clean rooms, storage spaces etc. Following are the features of the cascaded unit
[057] i) It facilitates chemical reactions requited for the disinfection of bacteria/fungus due to generated gases reactive species.

[058] ii) The process does not require consumable (chemical disinfectant),
however require only electrical energy and freely available air. [059] iii) The unit can be placed as a flooring tiles, in such a fashion that air
channel and electrical mesh electrode comes in contact. [060] iv) The tiles are connected with each other using silica putty for
insulating it from electrical discharge. [061] v) Air is circulated from the air channel, air goes through the channels,
through the upper metallic mesh electrode and then through perforated
tile to the atmosphere. [062] vi) Variable number of power supply units 10-50 kV, 20 - 100 Hz pulse
repetition frequency with 10-50 mA, can be used. [063] vii) Electrical timer unit that starts and stops after certain duration is
connected at the input.
[064] Example 2: Design of reactive species forming reactor assembly with miniaturized air-based dry sterilizer
[065] Figure 3 shows schematic of isometric view of the basic unit of prototype reactive species generator used to design cascaded unit shown in Figure 1 and Figure 2. The basic unit shown in Figure 3 is suitable for the disinfection of various microbial species as per the embodiment enclosed herewith. As seen from Figure 3 that the reactor chamber consists of 1) a box (reference no. 001, Figure 3) made up of acrylic having length ranging between 10-100 cm, breadth 10-50 cm and height 10 - 60 cm, which can be changed as per the requirements. 2) Inside the box a miniaturized air-based sterilizer unit (reference no. 002, Figure 3) having dimensions in the range of 8 - 80 cm length, 8 - 45 cm breadth and 8- 50 cm height, which can be changed as per the requirements, is mounted. Reference no. 003 to 012 in Figure 3 are power supply of the device (003), three pin power plug of the power supply (004), reactive species (plasma) distribution plate cum sample holder (005) having groves (006), top closing plate (007), holder for the top plate (008), air inlet (009), air out let (010) mesh having catalytic metal oxide coating (011), arrangement

to record optical emission spectra (012). For the detailed description of the miniaturized air-based sterilizer unit, refer Figure 4. Reference no. 13 to 22 in Figure 4 are Acrylic base plate (013, Figure 4) used to mount the unit, base plate of the bottom electrode (014, Figure 4), metallic (014 and 021) in Figure 4 and Figure 5) plate/mesh; one among the copper, aluminum, stainless steel or any other similar, sand-witched between two acrylic sheets of appropriate dimensions; dielectric separator (016, Figure 4) having holes drilled along X, Y (horizontal plane, 017, 018, Figure 4) and Z (vertical plane, 023, Figure 4) direction; stopper (019, Figure 4) provision kept to close the hole shown in (017 and 018) in Figure 4, if required; bottom plate of upper electrode (020, Figure 4); metallic mesh electrode with connecting electrode (021, Figure 4); upper plate of the top electrode (022, Figure 4). Figure 5 and Figure 6 show left and top view respectively of the device. The miniaturized air-based sterilizer unit consists of a metallic ((refer no. 014 and 021) in Figure 4 and Figure 5) plate/mesh; one among the copper, aluminum, stainless steel or any other similar, sand-witched between two acrylic sheets of appropriate dimensions. Pair of such assembly act as two electrodes. The pair of such electrodes is separated by means of spacer which keeps the separation of 1-10 mm between the two electrodes (ref. no. (016) in Figure 4 and Figure 5). Such type of one or multiple units may be mounted in a box. A pulsed DC power unit of 10-50 kV (20 - 100 Hz pulse repetition frequency) of dimension of about 10 cm x 10 cm x 5 cm is kept inside/outside of the box (no. (003) in Figure 3). Total weight of the power unit is about 500 g. There is provision of three pin electrical socket for each unit (no. (004) in Figure 3) to provide input power connection. Both, power supply and plasma forming unit are mounted onto the base plate of the box (no. (013) in Figure 4). Such one or multiple miniaturized air-based sterilizer are used. On the top of this unit an acrylic plasma distribution plate (ref. no. 005, Figure 3) of appropriate dimensions and grooves are drilled in it is mounted which facilitates propagation and

distribution of sufficiently long-lived reactive species. The top of the box shown in Figure 3 is closed with the help of another acrylic plate having appropriate dimensions and handle (ref. no. (007 and 008) in Figure 3). In addition, there is provision of a hole at the bottom to push air or other gases of interest in the reactive species forming unit (no. (009) in Figure 3) and at the top there is another hole (no. (010) in Figure 3), where from the resultant species comes out (no. (010) in Figure 3) through mesh having catalytic oxide coating on it (no. (011) in Figure 3). There is another hole to mount the optical fiber of an emission spectrometer (no. (012) in Figure 3).
During actual operation, the reactive species are generated along with UV-A and UV- B radiations. Following are some of the important plasma reactions during the reactive species formation:

[066] Example 3: Ozone detection:
[067] commercially, available Ozone detector (model, FORENSICS BH-90A (0-20ppm)) was used to detect the concentration of ozone at the place where the sample is kept as shown in (ref. no. (005), Figure 3). It is noted with the help of optical emission spectra, ozone is one of the major reactive species generated using the unit as discussed in example 2. The concentration of ozone detected by the detector is shown in Figure 7. The plot in Figure 7 indicates the ozone concentration increases drastically

within few 10 s seconds up to 20 ppm which is more than sufficient for
rapid disinfection of varieties of bacteria.
[068] Example 4: Miniaturized air-based sterilizer for antimicrobial study of E. Coli bacteria (Gram negative bacteria):
[069] The reactive species were generated using the experimental conditions
mentioned in example 2 and then the standard agar plates were used to
culture the E. Coli bacteria in conventional manner. The experiments were
planned as per the standard protocol where the optical density of the broth
was set to 0.18. The dilution was then adjusted to 10" . A drop of 50 µl of
the broth was then spread on each of 12 agar plates. The agar plates were
kept onto plasma (reactive species) distribution plate (ref no. (005) in
Figure 3) which was few cm away from miniaturized air-based sterilizer
and then sequentially exposed to reactive species generated as described in
example 2 and example 3 using an embodiment discussed, for the time
duration of a) 0 sec, b) 60 sec, c) 120 sec, d) 300 sec, e) 600 sec and f) 900
sec. Here the results of two set (total 12 samples) are included. After
treatment by reactive species all the plates were sealed with paraffin tape
and stored in an incubator in a controlled environment at 37 °C for 24 h.
The photographs of these plates recorded after 24 h of incubation are
shown in Figure 8. It is seen from the Figure 8 that exposure for the time
duration of 60 sec has inhibited the growth of E coli bacteria to large
extent. The counts of Colony Formation Unit (CFU) found to be dropped
down by 30- 60 % in case of the samples exposed for the duration of 60
sec. With increasing duration of exposure to 120 sec. the CFU count has
further reduced by 70-90 %. For the treatment duration of 300 sec, 600 sec
and 900 sec no CFU count were observed indicating the inhibition close to
100 %. Overall, the exposure time duration of 60 -120 sec seems to be
effective for the inhibition of the growth of E-Coli bacteria.
[070] Example 5: Miniaturized air-based sterilizer for antimicrobial study of Staphylococcus Auras bacteria (Gram positive bacteria)

[071] The experimental conditions used to inhibit the growth of Staphylococcus Auras bacteria (Gram positive bacteria) were same as discussed in example 4 for E-coli. Figure 9 is photograph of Staphylococcus Aureus grown in agar medium having initial concentration of 10" dilutions (0.18 OD) with treatment by reactive species generated using miniaturized air-based sterilizer for the duration of a) 0 sec, b) 60 sec. c) 120 sec. d) 180 sec, e) 300 sec and f) 600 sec. The photographs were recorded after 24 h of incubation at 37 °C. It is seen from Figure 9 that the plasma treatment for the duration of 60 sec drastically reduces CFU count of Staphylococcus Aureus.
[072] Example 6: Miniaturized air-based sterilizer for antimicrobial study of mixed bacteria collected from footwear:
[073] For the purpose to carry out disinfection of the mixed bacteria collected from footwear were used. The standard agar plates were prepared as mentioned earlier in example 4 and 5. Sterilized cotton swab was used to collect samples from the bottom surface of footwear. The swab samples so collected were dipped into 1 ml of saline and then 50 µl drop of such sample was spread over the agar plate. The agar plates so prepared were then exposed to reactive species generated using miniaturized air-based sterilizer as discussed in example 2 and 3 for the time duration of 0 - 120 sec. Finally, these samples were subjected to incubation for 24 h at 37 °C as per the standard protocol.
Figure 10 show photographs of CFU after 24 h of incubation period recorded for two set of samples. It is also seen that the different types of colonies have been formed.
It is clearly seen from the Figure 10 that exposure of the reactive species generated using miniaturized air-based sterilizer onto the sample for the duration of 30 sec reduces CFU significantly up to around 30 - 45 % compared to untreated plate. Whereas the CFU was reduced by 70-90% for the sample exposed to plasma species for the duration of 120 sec. Further increasing the time duration reduces the growth of the bacteria

more than 95 %. Thus, the reactive species so generated using the embodiment discussed in example 2 are effective to inhibit the growth of varieties of bacteria. [074] Applicability of the Miniaturized air-based sterilizer:
[075] From disclosure of this document, the utility and applicability of the unit can be extended to other devices based on same or modified plasma forming unit. As the unit can be fabricated in required form and dimensions to meet different requirements. The unit so formed can be fixed as tile of the floor and the reactive species concentration can be monitored by monitoring operating power, in put gas viz air, oxygen, nitrogen, H2O, H2O2 or combination of these reactive species having sufficiently long life time suitable for disinfection of varieties of bacteria relayed to respiratory system, dentistry or other bacteria, virus or fungus systems.
The various forms of the reactor unit are mouth mask, unit suitable for the disinfection of used mask/apron/gloves or any other components by the medical or other staff. In addition, it is possible to monitor concentration of various reactive species by varying the operating parameters and conditions.
[076] Based on the above-mentioned descriptions, the miniaturized air-based sterilizer so formed provides quick disinfection of the microbiological species with durability and long service life than any of its closest peers in state will be realized further. The present innovation is capable of various other embodiments and various alterations, all without departing from the basic concept of the present origination. Accordingly, as illustrative modifications will be obvious are intended to come within the territory of the present invention, which is limited only by the appended claims their localization and/or adaptations that may become necessary as part of description.

Claims:
We claim,
1] A method used for the inhibition of the growth of gram negative, gram positive as well as randomly mixed bacteria with the exposure to reactive species including ozone and other reactive species with the help of air-based miniaturized sterilizer. A portable, light weight, low power air-based sterilizer operated at the atmospheric pressure, temperature conditions, and an ambient air is used for the discharge. It does not require additional air or gas. However, to vary the concentration of desired reactive species, required gases such as air is introduced in the unit through an inlet, as one of the alternatives. This unit generates reactive species like OH, O, O*, N, N2 etc. along with ozone within a few second between two electrodes separated by dielectric medium. Further, these reactive species like O combines with O2 and forms O3 molecule. Based on the plasma chemistry, the formations of different reactive and molecular species are initiated once the device is activated. The effect of ozone/other reactive species on the bacterial/microorganism inhibition using the device is evident. The device comprises of:
• The reactive species generation unit which may consist of different number of basic units cascaded in a sequence, suitable as floor tile mount or wall mount.
• Air based reactive species generator along with power supply and other requirements enclosed in a box of perspex.
• The power supply used is very small in size, weight unlike bulky and expensive used in many cases. For the antimicrobial study, a sample of interest is kept onto the distribution plate (005); the fluence of the reactive species exposure has been decided based on the ON time of the device. Time of reactive species exposure varied in the range of 0 sec to 900 sec.

• The effects of air based reactive species on the bacterial inhibition after an exposure to the reactive species has been tested on isolated as well as randomly mixed bacteria, which is found to be effective.
2] A method used for inhibition of gram negative, gram positive as well as randomly mixed bacteria via exposure to the reactive species including ozone and other reactive species as discussed in example 3 and 4 The miniaturized air-based sterilizer unit consists of:
Acrylic base plate (013, Figure 4) used to mount the unit, base plate of the bottom electrode (014, Figure 4), metallic (refer no. (014 and 021) in Figure 4 and Figure 5) plate/mesh; one among the copper, aluminum, stainless steel or any other similar, sand-witched between two acrylic sheets of appropriate dimensions; dielectric separator (016, Figure 4) having holes drilled along X, Y (horizontal plane, 017, 018, Figure 4) and Z (vertical plane, 023, Figure 4) direction; stopper (019, Figure 4) provision used close the hole as shown (017 and 018) in Figure 4, if required; bottom plate of upper electrode (020, Figure 4); metallic mesh electrode with connecting electrode (021, Figure 4); upper plate of the top electrode (022, Figure 4).
• A pulsed DC power supply (003, Figure 4) of 10-50 kV (20 - 100
Hz pulse repetition frequency) of dimension of about 10 cm x 10
cm x 5 cm kept inside/outside the box is used to drive the reactive
species generation unit. Total weight of the power supply is about
500 g.
3] A method used for inhibition of gram negative, gram negative as well as mixed bacteria via exposure of reactive species so generated including ozone as claimed in example 2 and example 3, wherein the reactive species generator may be enclosed in a suitable encloser, such as:
• A parallelepiped box made up of perspex (001) of few mm
thicknesses and the volume 10 x 10 - 500 x 10 cm as described
in example 2.

• The removable reactive species distribution plate made up of perspex/acrylic (005) of appropriate dimensions, grooved thoroughly (006) having separation of few cm. This reactive species distribution plate was mounted few cm above the base of the perspex box (Figure 3 and Figure 4).
• An arrangement of exchange of specimen made through opening top plate (007) of area 102-104 cm2 , with an arrangement of handle (008) of appropriate dimensions.
• The reactive species generator (002) along with power supply (003) fixed inside the box (001). The power supply may be fixed outside the box. There is provision of three pin power plug for the power supply (004). Reactive species distribution plate is removable (005), which may be removed as and when required for further experiments.
4] The method of reactive species generation using the device as disclosed in the claims 1-3 is suitable for the generation of various reactive species few to name are atomic oxygen, molecular oxygen, ozone, N, N+, OH, N2 etc. of varied concentration.
5] The method of reactive species generation using the device as disclosed in the claims 1-3 is suitable for the inhibition of Gram negative bacteria e.g. E-coli that to more than 50 to 95 % with the exposure of reactive species for the duration of few tens to hundreds of sec having initial concentration with 10'2 dilutions (0.18 OD).
6] The method of reactive species generation using a device as disclosed in the claims 1- 3 is suitable for the inhibition of Gram positive bacteria e.g. Staphylococcus aureus to more the 50 to 95 % with the exposure of reactive species so generated for the duration of few tens to hundreds of sec. having initial concentration with 10" dilution (0.18 OD).
7] The method of reactive species generation using a device as disclosed in the claims 1-3 is suitable for the inhibition of randomly mixed bacteria collected from the footwear to more the 50 to 95 % with the exposure of

air based reactive species for the duration of few tens to hundreds of sec having initial concentration with 10" dilution (0.18 OD).
8] The miniaturized air-based sterilizer may be cascaded as shown in Figure 1 and Figure 2 to make it suitable as a floor mount/ wall mount wherever required. As per need the reactive species generator can be turned ON and OFF using suitable timer circuit so as to control the concentration/fluence of reactive species.
9] The method for generating reactive species of interest obtained using small, portable, rapid, low power operated, miniaturized air-based sterilizer for the inhibition of Gram negative, Gram positive as well as randomly mixed superbug required for medical applications as substantially illustrated in the accompanying description and drawings.

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