DESC:A DISPOSABLE ISOLATOR BAG FOR IN VIVO IMAGING OF SMALL ANIMALS IN A BIOCONTAINMENT FACILITY
The present subject matter relates to a disposable and inflatable transparent polyethylene-based biocontainment bag. In particular, the present subject matter discloses the disposable and inflatable transparent polyethylene-based biocontainment bag for imaging of live animals in microCT (or similar live animal imaging) instrument in Animal Biosafety Level 3 (ABSL3) or above laboratories.
BACKGROUND
Traditionally, in studies involving infectious agent that affects respiratory system or more specifically lungs, like involving SARS-CoV-2, CT scans are of utmost help and are a true predictive tool for the evaluation of efficacy of several therapeutic interventions. While the use of CT scans for variety of infectious disease model is an emerging field of research, however, the operationalization of these machines in an ABSL3 facility makes the whole thing very challenging and utterly complex. Several research groups have undertaken the use of microCT functions in ABSL3 facilities and characterized the use of different models and organ system studies. However, the containment practices while operating these instruments may be poorly followed, which is indicated by the lack of a suitable system or apparatus which can be used to image animals outside the biosafety cabinet in the imaging machines inside an ABSL3 facility.
Recently some groups have tried a highly regulated and functionally complex containment system which covers the whole imaging facility and restricts the chances of contamination. Such systems are quite complex and only avoid the contamination chances persisting during imaging. However, this does not address the check of contamination from infected animals transferred from biosafety cabinets to imaging systems and vice versa. Also, the containment facility has to undergo a quality control check in a span of few days which again is an extensive task and the sanitization process after each use is also an extra burden. Some groups have also tried with an animal isolation imaging chamber to maintain the containment in animal imaging, but the major drawback of this system is that it is not fully transparent from all the sides which is a prerequisite for X-ray based imaging and the box as such cannot get fit inside an X-ray based CT scanning machine animal bed. Even if this imaging chamber is made fully transparent or comes with an altered structure to be fitted in the CT machine beds, then the thickness of the material is likely to interfere in the imaging process or may reduce the quality of images post scanning. Moreover, because these systems can be used in a reusable manner so potential risk of breaking or scratching or even damage of the whole system persists and the repeated autoclave-based sanitization of these large units is not convenient for large number of animals. In addition to that, the autoclave-based sanitization or handling of these units may hamper the interest and speed of the user and may also elicit the scratching or breakage of these units.
Further, in ABSL-3 or higher facilities, while working with infected animals and performing imaging with live imaging platforms, two major problems arise. Those are, contamination risk to handlers or to animals not intended to get infected and secondly, the proper maintenance and monitoring of animals during the scanning time inside a containment apparatus or system.
There is a need for a solution to overcome above mentioned drawbacks.

SUMMARY
This summary may be provided to introduce concepts related to a disposable isolator bag used for in vivo imaging in a micro CT system; the concepts are further described below in the detailed description. This summary may be not intended to identify key features or essential features of the claimed subject matter, nor may be it intended to be used to limit the scope of the claimed subject matter.
The present subject matter provides a disposable isolator bag used for in vivo imaging in a micro CT system. The disposable isolator bag includes an inflatable enclosed chamber having a first side, a second side opposite to the first side, a base surface and an upper surface opposite to the base surface. The first side comprises an opening configured to allow an entry and an exit. The disposable isolator bag includes a plurality of holders projecting out of the upper surface from a first end and a second end for providing a holding means to the disposable isolator bag. The disposable isolator bag further includes a nozzle attached to the upper surface configured to receive air for inflating the inflatable enclosed chamber. The inflatable enclosed chamber is inflated with air to allow breathing inside the inflatable enclosed chamber.
In an embodiment of the present subject matter, the inflatable enclosed chamber is configured to receive an animal from the first side and the inflatable enclosed chamber is placed inside a CT scan machine for an isolated conduction of a CT scan of the animal.
In an embodiment of the present subject matter, each of the second side, the base surface and, the upper surface is permanently sealed to allow an entry and exit of an animal only via the first side.
In an embodiment of the present subject matter, the first side is directed towards a down flow HEPA-filtered air of a biosafety cabinet for inflating the inflatable enclosed chamber via the first side
In an embodiment of the present subject matter, the inflatable enclosed chamber is inflated after the animal is placed inside the inflatable enclosed chamber by pumping air into a nozzle orifice.
In an embodiment of the present subject matter, the nozzle comprises one or more valves to restrict a backflow of the air from an inside of the inflatable enclosed chamber to an outside.
In an embodiment of the present subject matter, the base surface comprises a plurality of demarcations to indicate a specific position on the base surface configured to receive the animal.
In an embodiment of the present subject matter, the first side is squeezed and tied with one of a rubber band, a clamp, a knot, a self-adhesive, a sealing machine, and a ribbon arrangement after the animal is placed inside theinflatable enclosed chamber.
In an embodiment of the present subject matter, the inflatable enclosed chamber is made up of polythene.
Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figs. In which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
The illustrated embodiments of the subject matter will be best understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description may be intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and methods that are consistent with the subject matter as claimed herein, wherein:
Fig. 1 illustrates a line diagram depicting a disposable isolator bag from the front view, in accordance with an embodiment of the present subject matter;
Fig. 2 illustrates a line diagram depicting the sedated animals inside a disposable isolator bag with a curved end and a flat end placed on a bed in a micro CT system, in accordance with an embodiment of the present subject matter;
Fig. 3a illustrates an image depicting representative CT scan images in coronal, axial and sagittal plane of hamster lungs of infected group and control with or without using the biocontainment bag, in accordance with an embodiment of the present subject matter; and
Fig. 3b illustrates a graphical representation depicting an analysis of CT images and quantification of aerated volume of the control lungs with or without using the biocontainment bag, in accordance with an embodiment of the present subject matter;
Fig. 4 illustrates a graphical representation depicting Ct (Cycle threshold) values of swab samples taken from infected hamsters’ oral cavity, from the interior of the biocontainment bag and exterior of the biocontainment bag after the scan, in accordance with an embodiment of the present subject matter; and
Fig. 5 illustrates a graphical representation depicting a survival percentage of hamsters scanned for ~6 mins with or without the biocontainment bag, in accordance with an embodiment of the present subject matter.

DETAILED DESCRIPTION
The following may be a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered may be not intended to limit the anticipated variations of embodiments; on the contrary, the intention may be to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure as defined by the appended claims.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
Fig. 1 illustrates a line diagram 100 depicting a disposable isolator bag in the front view 102 used for in vivo imaging in micro CT systems, in accordance with an embodiment of the present subject matter. The disposable isolator bag 102 may be used for a CT scan of a small animal that may be placed inside the disposable isolator bag 102 and the disposable isolator bag 102 may further be placed on a bed inside the micro CT system. Examples of the small animal may include, but are not limited to, mice, a hamster, a rat, a rabbit, and a guinea pig. The disposable isolator bag 102 may be a one-time use, flexible, highly contained, transparent thin system to seal infected animals from a surrounding environment but not affecting a survival of the infected animals while scanning without hampering scanning resolutions or image qualities. The infected animals may be sedated before placing in the disposable isolator bag 102 for the in vivo imaging of the infected animal in the microCT systems placed inside a biocontainment facility. The disposable isolator bag 102 may be a polyethene based isolator compatible for commercially available micro CT systems.
Continuing with the above embodiment, the disposable isolator bag 102 may include transparent (not affecting resolution, imaging quality and post scan image processing), highly contained (so that release of inside air is not allowed), leak proof, burst or damage proof provisions. The disposable isolator bag 102 may be used one time and disposed easily without requiring any sanitization for multiple usage. Further, the disposable isolator bag 102 may be inflated and may further be capable of holding enough air for a respiration of the sedated animal for a period of time. The period of time may at most range between ~6 minutes while scanning. The disposable isolator bag 102 may be flexible and compatible to sample beds of the micro CT system and the like.
To that understanding, the disposable isolator bag 102 may be compatible to a CT-scan machine bed and may provide a necessary respiratory comfort to the small animal while scanning and may also restrict a chance of spreading of infectious agents due to a leak-proof feature. Further, a body of the disposable isolator bag 102 may include a cylindrical inflatable transparent thin polythene sealed at three sides. At an opening side animals may be kept inside at desired place inside the disposable isolator bag 102 guided by numerical demarcations.
Continuing with the above embodiment, the disposable isolator bag 102 may include an inflatable enclosed chamber 104, a number of holders 106, and a nozzle 108. The inflatable enclosed chamber 104 may be configured to house the small animal while the disposable isolator bag 102 is placed on the bed in the micro CT system. The inflatable enclosed chamber 104 may be made up of polythene. Further, the inflatable enclosed chamber 104 may have a first side 104a, a second side 104b opposite to the first side 104a, a base surface 104d and an upper surface 104c opposite to the base surface 104d. The base surface 104d may include a number of demarcations to indicate a specific position on the base surface 104d configured to receive the animal. The demarcations printed on the base surface 104d may guide in placing the animal at a compatible place on the bed of the CT scan machine.
The first side 104a may include an opening configured to allow an entry and an exit of the small animal. The first side 104a may be squeezed and tied with one of a rubber band, a clamp, a knot, a self-adhesive, a sealing machine, and a ribbon arrangement after the small animal is placed inside the inflatable enclosed chamber 104. Furthermore, each of the second side 104b, the base surface 104d and, the upper surface 104c may be permanently sealed to allow an entry and exit of the small animal only via the first side 104a. After placing the animal inside, the first side 104a may be squeezed and tied. A volume of the disposable isolator bag 102 may have sufficient air to keep the sedated animals alive for a minimum time period of ~5-10 minutes.
Subsequently, the number of holder may be projecting out of the upper surface 104c from a first end and a second end of the upper surface 104c. The disposable isolator bag 102 may include at least two holders. The number of holders 106 may be configured to provide a holding means to the disposable isolator bag 102. The number of holders 106 may be projections from the disposable isolator bag 102 assisting in lifting the disposable isolator bag 102 and placing the disposable isolator bag 102 on the bed. Similarly, it may also be helpful in shifting the animals from the scanner to the biosafety hoods, after which the animals may be removed from the disposable isolator bag 102 and allowed for a recovery from anesthesia. The number of holders 106 is designed to keep on the either side of the numerical demarcation printed on the disposable isolator bag 102 to ensure that the number of holders 106 are not included during an animal scanning. The used isolators may be disposed of as per the SOP.
Furthermore, the nozzle 108 may be attached to the upper surface 104c. The nozzle 108 may be configured to receive air for inflating the inflatable enclosed chamber 104. The inflatable enclosed chamber 104 may be inflated with the air to allow breathing inside the inflatable enclosed chamber 104 when the small animal is inside the inflatable enclosed chamber 104 while the inflatable enclosed chamber 104 is placed inside the CT scan machine for an isolated conduction of a CT scan of the small animal. The nozzle 108 may include one or more valves to restrict a backflow of the air from an inside of the inflatable enclosed chamber 104 to an outside. The nozzle 108 may have a provision to inflate the disposable isolator bag 102 with a hand pump. After placing of the animals inside the disposable isolator bag 102 and sealing open ends the disposable isolator bag 102 may be inflated with hand pumps. Up to this process, each step should be performed under the biosafety hood. The fully inflated structure may also confirm absence of any leakage of air from the isolator.
In an embodiment of the present subject matter, the air may be received via the first side 104a. The first side 104a may be directed towards a down flow HEPA-filtered air of a biosafety for inflating the inflatable enclosed chamber 104 via the first side 104a. Also, the inflatable enclosed chamber 104 may be inflated after the small animal is placed inside the inflatable enclosed chamber 104 by pumping air into a nozzle orifice.
Fig. 2 illustrates a line diagram 200 depicting the disposable isolator bag 102 with a curved end and a flat end placed on a bed in a micro CT system, in accordance with an embodiment of the present subject matter. The disposable isolator bag 102 may include the inflatable enclosed chamber 104 having the curved end and the flat end. The inflatable enclosed chamber 104 may interchangeably be referred as an enclosed chamber. The enclosed chamber may be an inflatable, transparent housing sealed from at least three locations. At least one location may be an opening to allow entry of a living being such as sedated animal. In an example, an interior of the enclosed chamber may be provided with guides or demarcations to facilitate placement of the living being to be housed inside the chamber compatible to the CT scan beds. The opening is adapted to be sealed based on clamp, knot, band, self-adhesive, sealing machine or ribbon arrangement. The enclosed chamber may be inflated based on passing or releasing air via the nozzle 108. Inside the biosafety cabinet, the disposable isolator bag 102 may also be inflated by directing the open ends towards the down flow HEPA-filtered air followed by sealing by any of the aforementioned methods. The holder facilitates gripping of the enclosed chamber for transportation and portability.
Fig. 3a illustrates an image 300a depicting representative CT scan images in coronal, axial and sagittal plane of hamster lungs of infected animals with a biocontainment bag and control animals with or without the biocontainment bags, in accordance with an embodiment of the present subject matter. The control group of animal lungs may be imaged with or without the biocontainment bag. The biocontainment bag may be the disposable isolator bag 102 as referred in the fig. 1. No difference in resolutions of images taken with or without the bag is observed and animals scanned with the biocontainment bag displayed all typical features of a control lung through different sections of the lungs across different planes similar to the images obtained from the scanning of control lungs of a hamster without the use of biocontainment bags.
Furthermore, in the infected lungs, heavy consolidation may be observed around bronchi (red arrows) as a consequence of viral infection induced inflammation and pathophysiological changes in the lungs that may be properly visualized and monitored while using the disposable isolator bag 102.
Fig. 3b illustrates a graphical representation 300b depicting an analysis of CT images and quantification of aerated volume of the control lungs with or without using the biocontainment bag, in accordance with an embodiment of the present subject matter. Fig. 3b may further validate an absence of a technical hindrance in analyzing the CT images and also software defined quantification of aerated volume of the control lungs both with or without using the biocontainment bag and may further show a percentage of lungs volume with normal aerated and hypo-aerated/ consolidated areas for the said groups and conditions.
Fig. 4 illustrates a graphical representation 400 depicting Ct (Cycle threshold) values of swab samples taken from infected hamsters’ oral cavity from the interior of the biocontainment bag and exterior of the biocontainment bag after the scan, in accordance with an embodiment of the present subject matter. The Ct values from the oral swab may be in a range of detection, whereas the Ct values from the interior and exterior swab samples may not be in a detectable range i.e; Ct values >35.
Fig. 5 illustrates a graphical representation 500 depicting a survival percentage of hamsters scanned for ~6mins with or without the biocontainment bag, in accordance with an embodiment of the present subject matter. The result may depict that the biocontainment bag may not be intruding in the survival and breathing of animals during the time of scan. The biocontainment bag may be the disposable isolator bag 102 as referred in the fig. 1.
The disposable isolator bag 102 as disclosed in the present subject matter may have a number of uses as mentioned ahead. CT scan-based study for variety of infectious disease models is emerging with burgeoning interest and innovations, but in the meanwhile, the operationalization of these machines in an ABSL3 facility is a tough task looking at the contamination risk and functional difficulties in handling both infected animals and CT scan machines inside a BSL-3 facility. Initiatives have been adopted to use microCT in ABSL3 facilities to characterize the use of different models and organ system studies, however, the containment practice during animal scanning is not properly described which indicates the lack of a suitable system or apparatus which can be used to image animals outside the biosafety cabinet in the imaging machines inside an ABSL3 facility. This invention is likely to address the use of a novel polythene bases biocontainment bag design which can be readily used to isolate animals while performing live scanning, thus restricting the chance of spreading of malicious agents from infected animals, while putting considerable comfort to animals and ease to stay alive throughout the CT scanning procedure. This invention basically has three basic parts, first graduated body which may contain the sedated animals, second the nozzle 108 through which the body can be inflated and third the holders to hold and carry the bag. Thus, this design and the method cited here is novel, cost effective, has enormous commercial potential and may be a great contribution to the scientific community working with life threatening infectious agents in ABSL-3 or higher facilities.

This present subject matter addresses challenges in conducting a live CT based imaging of experimental animals in a ABSL3 facility without breaching containment measures. The polyethylene based disposable isolator bag 102 disclosed in the present subject matter compatible to CT-scan machine bed may provide the necessary respiratory requirements to animals while scanning and may also restrict a chance of spreading of infectious agents due to its leak-proof feature.
For using the disposable isolator bag 102, after anaesthesia the animal may be placed in the disposable isolator bag 102, after placing the animals, the disposable isolator bag 102 may be inflated with adequate air and may further be sealed under a biosafety cabinet hood. Graduation marks on a disposable isolator bag 102 body may guide a user to place the animal placed inside the disposable isolator bag 102 at correct positions, making it compatible to CT-scan machines and thus ensuring that a scanning process and a normal process of CT scanning may be followed.
The inflatable polybags also referred as the disposable isolator bag 102 may be featured with a nozzle 108 for air filling and holders for easy carry of the container having animals inside for imaging. Cylindrical bags which may be well fitted in a CT scan machine bed and a volume of the disposable isolator bag 102 may be so calculated to have adequate air for the easy breathing of animals during the whole process of scanning. Graduated disposable isolator bag 102 for a correct positioning of animals for scanning and for better customization with the scan machine. The disposable isolator bag 102 may be a disposable bag that may skip the sanitization process and safe to use for individual animals in biosafety level facilities. The disposable isolator bag 102 may be thin polyethylene based biocontainment bags providing necessary resistance to release of air present inside the disposable isolator bag 102 after inflation to the surrounding environment and thin polyethylene polymers may not hinder significantly the scanning image resolution and quality. The disposable isolator bag 102 may be configured to perform imaging of small animal models like hamsters, mice, rats etc. and ex vivo imaging of organs in experiments involving potential infectious agents or materials.
The disposable isolator bag 102 disclosed in the present subject matter may be used to image animals in BSL-2, BSL-3 and above facilities with other imaging platforms involving live animals and infection related works. The disposable isolator bag 102 may be used in a number of scenario explained in the form of examples below.

Examples
This may be referred to understand the use of the disclosure and also include the materials and methods adopted to ensure the proper functioning of the disclosure and fulfilment of all specifications explained in the innovation and claim sections. However, these examples can be considered for the reference of the procedures followed to check the potential and functioning of the disclosure and does not guarantee the experiments required to fulfil the claims of the disclosure and also does not specify the functioning of the disclosures or technical fulfilments of the claims. One can refer to these methods to understand the use of the said disclosure to check its efficiency or to use the disclosure in different experimental settings; however, this does not ensue the results of a particular experiment and does not limit the window of experiments that can be performed in a particular study.
Example-1: Leakproof test of the isolator
To check whether the isolator is leak proof or not we adopted bubble test. The inflated and sealed isolators were held under water and the tester watched for rising bubbles.
Example-2: SARS-CoV-2 infection of hamsters:
All the SARS-CoV-2 virus related experiments were carried out in the ABSL-3 facility and all the experiments were conducted after obtaining Institutional Biosafety Committee (IBSC) and Institutional Animal Ethical Committee (IAEC) approval. The virus, IND-ILS 01/2020 (Genbank accession ID- MW559533.2) was propagated in Vero-E6 cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% FBS and antibiotics. All the animals (aged 4-6 months) involved in this experiment were received from animal house and were shifted to maintained in IVCs at 25 ºC along with food and water provided ad libitum for 5 days prior to the start of the experiment to make them acclimatize to conditions prevailing therein. Animals were grouped into two groups: group 1; control group (animals not infected with virus) and group 2; infection group (animals challenged with SARS-CoV-2 virus). Three animals in each of these groups were housed in the ABSL-3 animal housing facilities for 10 days including 5 days of acclimatization and 5 days of experimental period. After completion of 5 days of acclimatization the control group animals were mock infected with PBS and the animals in the infected group were inoculated with SARS-CoV-2 virus intranasally. In advance of mock/virus infection animals were anaesthetized (with ketamine 200 mg/kg and xylazine 10 mg/kg per animal).The animals in the infection group were infected with 100 µl inoculum harboring ?10?^5 TCID50 of SARS-CoV-2 in PBS intranasally (50 µl per nostril). Animals in the control group were injected with 100µl PBS only. Extreme care was taken considering easy breathing of animals during infection and animals were monitored until they came back to senses.
Example-3: Sample collection:
Animals of the both the groups were imaged in the CT scan machine placed inside the ABSL-3 facility, one control animal was scanned without the use of the biocontainment polybag and the same animal from the same control group was scanned using the biocontainment polybag to compare, if there was any significant difference in the imaging clarity and resolution due to the use of the disclosure. Animals of the respective groups were sacrificed on 5 dpi and oral swab sample of animals were collected and swab sticks moistened with VTM were rubbed multiple times on different sites of different surfaces of the biocontainment bag which were in close contact with the animal during the imaging were also taken and immediately kept in -80?. Samples from different surfaces including inner and outer surface of the biocontainment polybag were used for quantification of viral RNA load.
Example-4: RNA isolation
Briefly all the tubes containing 1ml of VTM in which all the swab sticks were dipped during sample collections were vortexed. Then 200ul of the sample was used for RNA isolation. RNA was isolated using the Genolution VN143 Viral NA kit as per to the manufacturer’s instructions.
Example-5: cDNA synthesis and qRT PCR
cDNA was made with Applied Biosystems reverse transcription kit as per the manufacturer’s protocol. Briefly, same volume of RNA was picked from all the samples and mixed with the cDNA synthesis master mix to a total volume of 20 µl and the synthesis was done at 37? for 120 min using a thermocycler (eppendorf). RT PCR was performed by using the Promega SYBR green master mix and Ct value program in the QuantStudio6 real time PCR machine. Ct values above 35 were considered not detectable.
Example-6: CT scanning and analysis of hamster lungs:
Quantum GX-2 microCT imaging system (Perkin Elmer) was used for the micro CT based scanning of lungs of infected and control hamsters in the ABSL3 facility. Ketamine/xylazine anesthesia was used to sedate the experimental animals (hamster). The anaesthetized animals kept in a prostrate position in the sample bed of the micro CT machine during scanning and the position of the animal and internal structures were visualized in a scout view obtained through the Quantum GX2 software. The target organs for the experiment such as lungs and diaphragm were kept within a rectangular target box appeared in the software by manually moving the sample bed using machine commands. The animals correct position and the internal target organs proper orientation was examined at 90° and 180° and the scanning of the lungs was completed using QuantumGX2 software. For the scanning of the hamsters’ lungs Cu + Al (0.5 mm) X-ray filter was used with a 72mm field of views (FOVs) in a high resolution scan mode with 144 µm pixel size, 90kVp X-ray source voltage and 88µA current. The projection radiographs were taken throughout the 360° gantry rotation for a total scan time of 4 min and the raw files were saved as .vox files which were later analyzed using 2-D image slices obtained from the radiographs and with a 3-D reconstructed model using the analysis software.
Example-7: CT scan image analysis
The Analyze Direct 14.0 Software was used to analyse the data generated from the CT scans. The .vox files were uploaded to the Analyse 14.0 software and these lungs were subregioned. The subregioned files were fed in the process tool of the software, and a median correction with kernel size X:Y:Z ; 3:3:3 was performed which was further loaded in the segment tool in which the segmentation was carried out in a semi-automatic manner with the help of the object extractor tool. The segmented files obtained were further analyzed using the measure tool of the software, which resulted in obtaining the 3D volume of the aerated and consolidated/hypoaerated lungs.
Results
Leak proof biocontainment bag
A leak proof test may be conducted and validated through the bubble test. No air bubbles may be noticed after inflating the biocontainment bags to the maximum extent in standard conditions during the experimental period, which suggests that the isolators may be totally airtight and spillage proof.

CT scan analysis of the hamster lungs with and without the biocontainment bag
CT scan analysis of control hamster’s thoracic area may be performed with or without the biocontainment bag/isolator. There may be no difference in the resolution of the scanned images (Fig.03a). As per the expectation, CT scan images showed larger consolidated/ hypo-aerated areas in infected animal’s lungs compared to non-infected animals indicating presence of heavy infection and pathophysiological events associated with SARS-CoV-2 in infected animals lungs which may be imaged after being put inside the biocontainment bag. Quantification of the hypo-aerated lung volume also corroborated these findings (Fig.03b). Together, the biocontainment bag does not limit the experimental output. Again, no difference in the aerated volume of the control group’s lungs may be obtained both in presence and absence of the biocontainment bag indicating the use of the biocontainment bags did not limit the technical components of the CT scan process and neither the CT scan resulted radiographs nor the software defined quantification of lung aerated volume had any difference in the presence or absence of the biocontainment bag.

qRT PCR analysis
The Ct values of the swab samples taken from both inside and outside of the biocontainment bag reveals the absence of viral contamination from the infected animals, as the Ct values of the sample taken from outside the bag may be not in the detectable range (Fig.04). At the same time, viral genome presence may be detected in the oral swabs of the infected animals. Hence, the biocontainment bags are able to restrict the spread of contaminating agents.

Effect on the survival of the animal
The biocontainment polybag does not affect survival of the animals during CT-scanning and all the animals imaged using the biocontainment bags survived getting past the anaesthesia even in the infected group showing no visible signs of stress or illness (Fig.05).

Biocontainment bags offer no respiratory distress to the animals during the scan time The animals kept inside biocontainment bags and also during imaging under the CT scan machine showed no visible respiratory discomfort. Animals kept within or without biocontainment bags showed equal breathing rate and normal breathing may be observed and no forced/abdominal breathing may be observed manually or during the CT scanning as observed under the scout view.
Industrial application
The present disclosure may be used in all biosafety level facilities including BSL2, BSL-3 or above facilities and this may require huge production of this polybags (isolators) to ensure rapid supplies to all labs and companies around the globe who use contaminating pathogens as a disease models with a potential threat of direct or indirect contamination, use varieties of imaging facilities including CT scan, which cannot be operated inside a biosafety cabinet and may opt this biocontainment polybag for imaging related experiments.
While the detailed description describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention may be determined by the claims that follow. The invention may be not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
,CLAIMS:We claim:
1. A disposable isolator bag (102) used for in vivo imaging in a micro CT system, comprising:
an inflatable enclosed chamber (104) having a first side (104a), a second side (104b) opposite to the first side (104a), a base surface (104d) and an upper surface (104c) opposite to the base surface (104d), wherein the first side (104a) comprises an opening configured to allow an entry and an exit;

a plurality of holders (106) projecting out of the upper surface (104c) from a first end and a second end for providing a holding means to the disposable isolator bag; and

a nozzle (108) attached to the upper surface (104c) configured to receive air for inflating the inflatable enclosed chamber, wherein the inflatable enclosed chamber (104) is inflated with air to allow breathing inside the inflatable enclosed chamber.

2. The disposable isolator bag (102) as claimed in claim 1, wherein the inflatable enclosed chamber (104) is configured to receive an animal from the first side and the inflatable enclosed chamber (104) is placed inside a CT scan machine for an isolated conduction of a CT scan of the animal.
3. The disposable isolator bag (102) as claimed in claim 1, wherein each of the second side (104b), the base surface (104d) and, the upper surface (104c) is permanently sealed to allow an entry and exit of an animal only via the first side (104a).
4. The disposable isolator bag (102) as claimed in claim 1, wherein the first side (104a) is directed towards a down flow HEPA-filtered air of a biosafety cabinet for inflating the inflatable enclosed chamber (104) via the first side (104a).
5. The disposable isolator bag (102) as claimed in claim 1, wherein the inflatable enclosed chamber (104) is inflated after the animal is placed inside the inflatable enclosed chamber by pumping air into a nozzle orifice.
6. The disposable isolator bag (102) as claimed in claim 1, wherein the nozzle (108) comprises one or more valves to restrict a backflow of the air from an inside of the inflatable enclosed chamber (104) to an outside.
7. The disposable isolator bag (102) as claimed in claim 1, wherein the base surface (104d) comprises a plurality of demarcations to indicate a specific position on the base surface (104d) configured to receive the animal.
8. The disposable isolator bag (102) as claimed in claim 1, wherein first side (104a) is squeezed and tied with one of a rubber band, a clamp, a knot, a self-adhesive, a sealing machine, and a ribbon arrangement after the animal is placed inside the inflatable enclosed chamber.
9. The disposable isolator bag (102) as claimed in claim 1, wherein the inflatable enclosed chamber (104) is made up of polythene.