FIELD OF THE INVENTION
[001] The present invention relates to the field of anaerobic culture of microorganisms and, more particularly, the present invention relates to the oxygen-scavenging Petri dish and composition for growing anaerobic microorganisms. More particularly, the present disclosure relates in a general way to growing anaerobic microbial cultures on a petri dish having Petri dish consisting of lid and base of the dish.
BACKGROUND FOR THE INVENTION:
[002] The following discussion of the background of the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known, or part of the common general knowledge in any jurisdiction as of the priority date of the application. The details provided herein the background if belongs to any publication is taken only as a reference for describing the problems, in general terminologies or principles or both of science and technology in the associated prior art.
[003] Petri dish is used to grow microorganisms (Osman Erkmen, Laboratory Practices in Microbiology, 1st Edition, 2021, ISBN: 9780323910170). The Petri dishes contain two plates, a receptacle, and an additional lid that fits over the bottom one. The petri dish is filled with a microbial culture medium for the growth of microorganisms and cells. Evenly spread microbial culture on a petri dish enables the isolation of an individual microorganism for its further identification and utility. Microorganisms are divided into four major groups namely aerobic which require oxygen, facultative anaerobes which can grow with or without oxygen, microaerobic, which can grow only in very low levels of oxygen, and anaerobes which cannot grow in the presence of oxygen. Various design of Petri dishes is available to grow such microorganisms. The present invention is directed to the development of an anaerobic cultivation system of a Petri dish with improved properties with respect to the enhanced ability to use it with increased efficiency which is required to culture anaerobes microorganisms. Such microbes will be of immense applied value for food, biotechnological, pharmaceutical, and environmental industries. To ensure whether an anaerobic atmosphere exists in the jar, an indicator strip of methylene blue (oxygen indicator) is used that becomes colorless in absence of oxygen.
[004] U.S. Pat. No. 11,286,450 relates to culture systems and apparatuses which can be used to cultivate microorganisms in a low-oxygen environment.US patent 2,825,651 invention relates to an in-package oxygen remover for packaged dry products which are susceptible to deterioration due to the presence of oxygen. Anhydrous sodium sulfite and copper sulfate pentahydrate are brought into imminent contact, either by grinding them together or by compressing them into a pellet, to trigger the reaction to serve as an effective oxygen remover.US patent 3,165,450 describes the removal of residual oxygen for the sealed petri dish by the reaction of the pyrogallic acid and sodium carbonate to induce culturing of the anaerobic microorganism.US patent. No. 2005.0034599A1 describes an oxygen absorber agent composition comprising 100 parts by weight of iron powder and 0.1 parts by weight or more of a promoter comprising an alkali metal salt or alkali earth metal Salt having low moisture transportation properties.
[005] U S patent no. 10, 450, 244 B2 relates to a method for the removal of oxygen from hydrogen using manganese, iron, nickel, or cobalt-based adsorbent, without the generation of water or other oxides.U S patent no. 11,072,771 describes a Self-contained anaerobic culture device with micro compartments that facilitate the growth of an anaerobic microorganism by partitioning the sample into aliquots so that the desired reaction or growth occurs and can be detected much more rapidly than the same reaction or growth in the original larger volume. It also has the ability to detect an indication of the growth of the anaerobic microorganism using an oxygen-scavenging reagent selected from the group consisting of ferrous and ascorbic acid and salts thereof.
[006] In light of the foregoing, there is a need for the Oxygen scavenging Petri dish and composition for growing anaerobic microorganisms that overcome problems prevalent in the prior art.
OBJECTS OF THE INVENTION:
[007] Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
[008] The principal object of the present invention is to overcome the disadvantages of the prior art by providing the oxygen scavenging Petri dish and composition for growing anaerobic microorganisms that simply and quickly provides a controlled environment suitable for growing and maintaining anaerobic microorganisms useful in healthcare, agriculture and industry.
[009] An object of the present invention is to provide an oxygen scavenging Petri dish and composition for growing anaerobic microorganisms, wherein the Petri dish has utility for growing the anaerobic microbial cultures.
[010] It is an object of the present invention to provide an oxygen absorbing composition for use as a packaging material that absorbs oxygen quickly.
[011] Another object of the present invention is to provide an oxygen scavenging Petri dish and composition for use as a component of resin packaging material that is relatively simple and is used to create an anaerobic condition quickly in any vessel, for example petri dish but not limited to it.
[012] Another object of the present invention is to provide an oxygen scavenging Petri dish and composition for growing anaerobic microorganisms that provides an oxygen indicator strip that changes color as per availability of oxygen, to ensure that an anaerobic atmosphere exists in the Petri dish with a culture media placed within the container.
[013] Another object of the present invention is to provide the oxygen scavenging Petri dish and composition for growing anaerobic microorganisms that provides an effective synergistic effect of the magnesium chloride on anaerobiosis among all the chloride salts tested.
[014] Another object of the present invention is to provide the oxygen scavenging Petri dish and composition for growing anaerobic microorganisms that provides an oxygen-absorbing composition in order to carry out the absorption of oxygen.
[015] Yet another object of the present invention is to provide the oxygen scavenging Petri dish and composition for growing anaerobic microorganisms that is useful for manipulating a microbial sample in a laboratory or other controlled environment.
[016] Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION:
[017] The present invention provides an oxygen-scavenging Petri dish and composition for growing anaerobic microorganisms.
[018] According to one aspect of the present invention, the Petri dish comprises a base, a sidewall extending from the base, and the lid comprises a central horizontal wall and a top wall. The lid falls relative to the base of the dish when the lid is pressed it snugly fits in the rubber ring lining on the outer circumference of the base, and the petri dish is sealed tightly, to exclude ambient air.
[019] Further to ensure that the residual air left in the petri dish does not contain oxygen the upper lid of the petri dish contains about 10 gm. of oxygen-scavenging material a novel Swami Rama Himalayan University-anaerobiosis formulation (SRHU-AN) in about 9.8 gm. comprising ascorbic acid, 1.24 gm., cobalt chloride, 1.24 gm., sodium bicarbonate, 2.48 gm., ferrous carbonate, 2.48 gm., activated carbon, 2.24, and magnesium chloride, 0.124 gm kept in a transparent polyurethane material which is permeable to gases and has an adhesive backing attached to the inner side of the top lid in a chamber, sealed with a cover, which can be peeled off. To initiate the oxidation reaction oxygen absorbent composition is injected with sterile water. Status of the anaerobic conditions within the sealed petri dish inoculated with microorganisms is demonstrated by an oxygen indicator pink color of resazurin dye to colorless, due to the change in colour.
BRIEF DESCRIPTION OF DRAWINGS:
[020] Reference will be made to embodiments of the invention, examples of which may be illustrated in accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in the context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
[021] Figure 1 shows an isometric assembly in a wireframe view of a petri dish, according to an exemplary embodiment of the present invention;
[022] Figure 2 shows an isometric assembly view of one embodiment of the apparatus of the tool of Figure 1;
[023] Figure 3 shows a cross-sectional assembly view of the upper and lower Petri plate showing removable strips with fine grain meshed sheet with an oxygen absorber in a canister and dye indicator on the upper plate of the Petri plate; and
[024] Figure 4 is a view of the lower part of the petri dish with a silicone rubber ring to ensure that an anaerobic atmosphere exists in the Petri dish.
DETAILED DESCRIPTION OF DRAWINGS:
[025] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and is not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.
[026] As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.
[027] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.
[028] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.
[029] The present invention provides Oxygen scavenging Petri dish (100) and composition for growing anaerobic microorganisms.
[030] the present invention describes a petri dish (100)comprising a base, a sidewall extending from the base, and a track extending outwardly from the outer surface of the sidewall. The lid comprises a central horizontal wall and a recess formed by two sidewalls and a top wall. The lid falls relative to the base of the dish when the lid is pressed it snugly fits in the rubber ring lining on the outer circumference of the base, and the petri dish (100)is sealed tightly. This creates a hermetic seal at the lip of the petri dish(100) thereby excluding ambient air. Further to ensure that the residual air left in the petri dish (100)does not contain oxygen for the optimal growth of anaerobic microorganisms, the design of the petri dish(100)has been changed in such a manner that the upper lid of the petri dish contains oxygen-scavenging materials. Once the reaction has started and anaerobic conditions have been generated the growth of anaerobic microorganisms is demonstrated by an oxygen indicator (5)due to a change in color.
[031] The invention is based upon the design which keeps oxidizable components consisting of an oxygen scavenging system separate in a container, till the time of oxygen scavenging is required. The oxidizable component is kept in a container with a covering laminated film which can be peeled off to allow access to the packaged contents having a bag shape formed by an air-permeable to initiate the reaction by the oxidation catalyst. Oxidizable components consisting of an oxygen scavenging system are contemplated to be in any size or shape including sachet, pellets, pouch, strip, and cartridge, etc. Oxygen absorber consisting of about 9.8 gm. comprising ascorbic acid, 1.24 gm., cobalt chloride, 1.24 gm., sodium bicarbonate, 2.48 gm., ferrous carbonate, 2.48 gm., activated carbon, 2.24, and magnesium chloride, 0.124 gm were weighed and mixed to produce an oxygen absorbent (4) composition. The 10.0 g deoxygenation agent thus prepared is filled in a transparent polyurethane material which is permeable to gases and has an adhesive backing. An indicator strip(5)of pink-colored resazurin dye is used that becomes colorless in absence of oxygen. Sterilization of the petri dish(100) so prepared is preferred by using gamma radiation. Once the lower (5) lid of the petri dish(100)is inoculated with microorganisms, the upper lid of the petri dish (100)containing the oxygen absorbent(4) is injected with adding 3.0 ml sterile water, by peeling off the seal. Oxygen-scavenging materials generate H2 and CO2 when water is added. H combines with O2 in presence of a catalyst. Immediately afterward the lid is pressed to create a hermetic seal of the petri dish (100)to exclude ambient air. Change in the pink color of resazurin dye to colorless indicates the achievement of the anaerobic status of the petri dish(100), a conducive environment for the growth of anaerobic microorganisms.
[032] With reference now to the drawings, Figures 1 to 4 thereof, a novel method and an apparatus of Petri dishes(100)have been described, embodying the principles and concepts of the disclosed subject matter will be described.
[033] Petri dish(100)comprises a base, a sidewall extending from the base, and the lid comprises a central horizontal wall and a top wall. The lid falls relative to the base of the dish when the lid is pressed it snugly fits in the rubber ring(7) lining on the outer circumference of the base, and the petri dish(100)is sealed tightly, to exclude ambient air.
[034] Further to ensure that the residual air left in the petri dish(100) does not contain oxygen the upper lid of the petri dish(100) contains about 10 gm. of oxygen-scavenging material a novel Swami Rama Himalayan University-anaerobiosis formulation (SRHU-AN) in about 9.8 gm. comprising ascorbic acid, 1.24 gm., cobalt chloride, 1.24 gm., sodium bicarbonate, 2.48 gm., ferrous carbonate, 2.48 gm., activated carbon, 2.24, and magnesium chloride, 0.124 gm kept in a transparent polyurethane material which is permeable to gases and has an adhesive backing attached to the inner side of the top lid in a chamber, sealed with a cover, which can be peeled off. To initiate the oxidation reaction oxygen absorbent(4) composition is injected with sterile water. The status of the anaerobic conditions within the sealed petri dish(100) inoculated with microorganisms is demonstrated by an oxygen indicator(5)pink color of resazurin dye to colorless, due to change in color.
[035] An important purpose of the apparatus is to simply and quickly provide a controlled environment suitable for growing and maintaining anaerobic microorganisms useful in healthcare, agriculture, and industry.
[036] In a preferred composition, the present invention provides an oxygen-absorbing composition in order to carry out the absorption of oxygen by means of which it comprises is present by weight in an amount between a combination of ascorbic acid 1% and 40% and most preferably between 10% and 20%, cobalt chloride 1% and 40% and most preferably between 10% and 20%, sodium bicarbonate 5% and 80% and most preferably between 15% and 40%, ferrous carbonate 5% and 80% and most preferably between 15% and 40%, activated carbon 5% and 80% and most preferably between 15% and 40%, and magnesium chloride 0.1% and 20% and most preferably between 0.5% and 4%.
[037] While ascorbic acid, cobalt chloride, sodium bicarbonate, ferrous carbonate, activated carbon, and magnesium chloride, are the preferred salts to produce an oxygen-absorbent(4) composition, other salts are not limited to ascorbic acid, cobalt chloride, sodium bicarbonate, ferrous carbonate, activated carbon, and magnesium chloride and can be used that work in a satisfactory manner.
[038] In various aspects, the disclosure relates to embodiments of an apparatus of growing microbial cultures that may be useful for manipulating a microbial sample in a laboratory or other controlled environment.
[039] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example 1: Formulations for anaerobiosis
[040] Oxygen absorber consisting of ascorbic acid, cobalt chloride, sodium bicarbonate, ferrous carbonate, and activated carbon were weighed and mixed in following 4 configurations A, B, C, D to produce an oxygen absorbent(4) composition for anaerobiosis.
A B C D
Ascorbic acid 1 2 3 4
Cobalt chloride 2 2 2 2
Sodium bicarbonate 4 4 4 4
Ferrous carbonate 2 4 6 8
Activated carbon 2 2 2 2
[041] Thus, the following composition in gm. was used as an example for preparing about 10 gm. mixture for anaerobiosis. Once the lower (5)lid of the petri dish(100) is inoculated with microorganism, upper lid of the petri dish (100)containing the oxygen-absorbent(4) mixture was injected with 3.0 ml sterile water, by peeling off the seal(7). Oxygen-scavenging materials generate H2 and CO2 when water is added. H combines with O2 in presence of a catalyst. Immediately afterward the lid is pressed to create a hermetic seal(7) of the petri dish (100)to exclude ambient air. The change in the pink color of resazurin dye to colorless indicates the achievement of the anaerobic status of the petri dish(100), a conducive environment for the growth of anaerobic microorganisms. The time taken by the formulations A, B, C, and D of the pink color of resazurin dye to colorless was 2 h, 15 min., 1 h 40 min, and 2 h 30 min, respectively. Thus, formulation B was selected for further work.
A B C D
Ascorbic acid 0.76 1.24 1.56 1.76
Cobalt chloride 1.54 1.24 1.04 0.88
Sodium bicarbonate 3.04 2.48 2.08 1.76
Ferrous carbonate 1.54 2.48 3.12 3.52
Activated carbon 1.54 1.24 1.04 0.88
Time 2 h 15 min 1 h 40 min 2 h 30 min
Example 2: Effect of ascorbic acid on anaerobiosis
[042] The time taken by the formulations B with 100% ascorbic acid 1.24 gm, set 1 with 50% ascorbic acid 0.62 gm., and Set 2 with 10% ascorbic acid 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 12 min., 34 min., and 47 min, respectively. Thus 1.24 gm ascorbic acid was selected for further work.
B (standard) Set 1 Set 2
Ascorbic acid 1.24 (100 %) 0.62 (50 %) 0.124 (10 %)
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Time 12 min 34 min 47 min
Example 3: Effect of cobalt chloride on anaerobiosis
[043] The time taken by the formulations B with 100% ascorbic acid 1.24 gm, Set 1 with 50% cobalt chloride 0.62 gm., and Set 2 with 10% cobalt chloride 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 14 min., 36 min., and 55 min, respectively. Thus 1.24 gm of cobalt chloride was selected for further work.
B (standard) Set 1 Set 2
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 (100%) 0.62 (50 %) 0.124 (10 %)
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Time 14 min 36 min 55 min
Example 4: Effect of sodium bicarbonate on anaerobiosis
[044] The time taken by the formulations B with 100% sodium bicarbonate 2.48 gm, Set 1 with 50% sodium bicarbonate 1.24 gm., and Set 2 with 10% sodium bicarbonate 0.248 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 17 min., 40 min., and 58 min., respectively. Thus 2.48 gm sodium bicarbonate was selected for further work.
B (standard) Set 1 Set 2
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 (100%) 1.24 (50 %) 0.248 (10 %)
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Time 17 min 40 min 58 min
Example 5: Effect of ferrous carbonate on anaerobiosis
[045] The time taken by the formulations B with 100% ferrous carbonate 2.48 gm, set 1 with 50% ferrous carbonate 1.24 gm., and Set 2 with 10% ferrous carbonate 0.248 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 15 min., 38 min., and 50 min., respectively. Thus 2.48 gm ferrous carbonate was selected for further work.
B (standard) Set 1 Set 2
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 (100%) 1.24 (50%) 0.248 (10%)
Activated carbon 1.24 1.24 1.24
Time 15 min 38 min 50 min
Example 6: Effect of activated carbon on anaerobiosis
[046] The time taken by the formulations B with 100% activated carbon 1.24 gm, Set 1 with 50% activated carbon 0.62 gm., and Set 2 with 10% activated carbon 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 12 min., 29 min., and 40 min., respectively. This 1.24 gm activated carbon was selected for further work.
B (standard) Set 1 Set 2
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 12 min 29 min 40 min
Example 7: Effect of additional chloride, ferric chloride, on anaerobiosis
[047] The time taken by the formulations B, set 1 with 100% ferric chloride 1.24 gm, Set 2 with 50% ferric chloride 0.62 gm., and Set 3 with 10% ferric chloride 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 1 min 45 sec., 1 min 35 sec., and 1 min 21 sec., respectively.
Set 1 Set 2 Set 3
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Ferric chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 1 min 45 sec 1 min 35 sec 1 min 21 sec
Example 8: Effect of additional chloride, ammonium chloride, on anaerobiosis
[048] The time taken by the formulation B, Set 1 with 100% ammonium chloride 1.24 gm, Set 2 with 50% ammonium chloride 0.62 gm., and Set 3 with 10% ammonium chloride 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 2 min 50., 2 min 6 sec., and 1 min 10 sec., respectively.
Set 1 Set 2 Set 3
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Ammonium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 2 min 50 sec 2 min 6 sec 1 min 10 sec
Example 9: Effect of additional chloride, sodium chloride, on anaerobiosis
[049] The time taken by formulation B, Set 1 with 100% sodium chloride 1.24 gm, Set 2 with 50% sodium chloride 0.62 gm., and Set 3 with 10% sodium chloride 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 4 min 45 sec., 4 min 2 sec., and 3 min., respectively.
Set 1 Set 2 Set 3
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Sodium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 4 min 45 sec 4 min 2 sec 3 min
Example 10: Effect of additional chloride, calcium chloride, on anaerobiosis
[050] The time taken by formulation B, Set 1 with 100% calcium chloride 1.24 gm, Set 2 with 50% calcium chloride 0.62 gm., and Set 3 with 10% calcium chloride 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 1 min 20 sec., 1 min 19 sec., and 1 min 20 sec., respectively.
Set 1 Set 2 Set 3
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Calcium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 1 min 20 sec 1 min 19 sec 1 min 20 sec
Example 11: Effect of additional chloride, magnesium chloride, on anaerobiosis
[051] The time taken by formulation B, Set 1 with 100% magnesium chloride 1.24 gm, Set 2 with 50% magnesium chloride 0.62 gm., and Set 3 with 10% magnesium chloride 0.124 gm., while keeping other components constant, of the pink color of resazurin dye to colorless was 45 sec., 40 sec., and 42 sec., respectively. This 0.124 gm magnesium chloride was selected for further work.
Set 1 Set 2 Set 3
(SRHU-AN)
Ascorbic acid 1.24 1.24 1.24
Cobalt chloride 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Magnesium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 45 sec 40 sec 42 sec

[052] This novel Swami Rama Himalayan University-anaerobiosis formulation (SRHU-AN) in about 9.8 gm. consisted of ascorbic acid, 1.24 gm., cobalt chloride, 1.24 gm., sodium bicarbonate, 2.48 gm., ferrous carbonate, 2.48 gm., activated carbon, 2.24, and magnesium chloride, 0.124 gm, was thus used for further studies. To initiate the anaerobiosis reaction 3 ml. the water was added.
Example 12: Effect of different chloride salts (gm) concentrations by the formulation B in the absence of cobalt chloride on anaerobiosis
[053] The time is taken by the formulation B in the presence of different chloride salts ferric chloride, ammonium chloride, sodium chloride, calcium chloride, and magnesium chloride individually, in the absence of cobalt chloride, on anaerobiosis was elucidated, with 100% salt 1.24 gm, Set 1 with 50% salt 0.62 gm., and Set 2 with 10% salt 0.124 gm., while keeping other components being constant. The minimum time taken to change the pink color of resazurin dye to colorless was 3 min 25 sec., by using 1.24 gm magnesium chloride. Among all the chloride salts tested magnesium chloride demonstrated the most effective synergistic activity of anaerobiosis, as demonstrated previously in Example 11, Set 3.
Components Set 1 Set 2 Set 3
Ascorbic acid 1.24 1.24 1.24
Sodium bicarbonate 2.48 2.48 2.48
Ferrous carbonate 2.48 2.48 2.48
Activated carbon 1.24 1.24 1.24
Ferric chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 6 min 30 sec 7 min 45 sec 9 min
Ammonium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 8 min 15 sec 8 min 45 sec 10 min 30 sec
Sodium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 10 min 12 min 20 sec 15 min 25 sec
Calcium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 4 min 15 sec 5 min 40 sec 7 min 35 sec
Magnesium chloride 1.24 (100%) 0.62 (50%) 0.124 (10%)
Time 3 min 25 sec 4 min 50 sec 6 min 10 sec

[054] The results further validated the most effective synergistic effect of magnesium chloride on anaerobiosis among all the chloride salts tested, which construes formulation B, as demonstrated in Example 11, Set 3 by SRHU-AN.
[055] Following table provides comparative data for demonstrating the synergistic effect of magnesium chloride, defined in terms of the minimal time taken to scavenge the oxygen.
S. No Ascorbic acid Cobalt chloride Sodium bicarbonate Ferrous carbonate Activated carbon Time
1 1.24* 1.24 2.48 2.48 1.24 15 min
2 0.62** 1.24 2.48 2.48 1.24 34 min
3 0.124*** 1.24 2.48 2.48 1.24 47 min
4 1.24 0.62** 2.48 2.48 1.24 36 min
5 1.24 0.124*** 2.48 2.48 1.24 55 min
6 1.24 1.24 1.24** 2.48 1.24 40 min
7 1.24 1.24 0.248*** 2.48 1.24 58 min
8 1.24 1.24 2.48 1.24** 1.24 38 min
9 1.24 1.24 2.48 0.248*** 1.24 50 min
10 1.24 1.24 2.48 2.48 0.62** 29 min
11 1.24 1.24 2.48 2.48 0.124*** 40 min
Ferric chloride
12 1.24 1.24 2.48 2.48 1.24 1.24* 1 min 45 sec
13 1.24 1.24 2.48 2.48 1.24 0.62** 1 min 35 sec
14 1.24 1.24 2.48 2.48 1.24 0.124*** 1 min 21 sec
Ammonium chloride
15 1.24 1.24 2.48 2.48 1.24 1.24* 1 min 50 sec
16 1.24 1.24 2.48 2.48 1.24 0.62** 2 min 06 sec
17 1.24 1.24 2.48 2.48 1.24 0.124*** 1 min 10 sec
Sodium chloride
18 1.24 1.24 2.48 2.48 1.24 1.24* 4 min 45 sec
19 1.24 1.24 2.48 2.48 1.24 0.62** 4 min 02 sec
20 1.24 1.24 2.48 2.48 1.24 0.124*** 3 min
Calcium chloride
21 1.24 1.24 2.48 2.48 1.24 1.24* 1 min 20 sec
22 1.24 1.24 2.48 2.48 1.24 0.62** 1 min 19 sec
23 1.24 1.24 2.48 2.48 1.24 0.124*** 1 min 20 sec
Magnesium chloride
24 1.24 1.24 2.48 2.48 1.24 1.24* 45 sec
25 1.24 1.24 2.48 2.48 1.24 0.62** 40 sec
26 1.24 1.24 2.48 2.48 1.24 0.124*** 42 sec
[056] The disclosure has been described withreference to the accompanying embodiments herein and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein.
[057] The foregoing description of the specific embodiments so fully revealed the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described herein.

We Claim:

1) An oxygen-scavenging petri dish apparatus (100), the apparatus comprising:a lid having an oxygen-scavenging absorbent (4) and oxygen-indicator (5)member;a dish having a base;when falls relative to the base of the dish when the lid is pressed, snugly fits in a rubber ring (7) lining on an outer circumference of the base, a stopper with respect to the atmosphere outside the container; andwhen a fastener-end member is fully engaged with the petri dish (100) is a hermetically sealed oxygen barrier to grow anaerobic microorganisms;
wherein theoxygen-scavenging absorbent (4) is consisting of 10% - 20% of ascorbic acid, 10% - 20% of cobalt chloride, 15% - 40% of sodium bicarbonate, 15% - 40% of ferrous carbonate, 15% - 40% of activated carbon, and 0.5% - 4% of magnesium chloride by weight.
2) The apparatus(100)as claimed in claim 1, wherein the absorbent (4) is packaged within a gas-permeable material, which has an adhesive backing attached to the inner side of a top lid in a chamber, sealed with a cover.
3) The apparatus (100)as claimed in claim 2, wherein the adhesive backing is detachably attached.
4) The apparatus (100)as claimed in claim 1, wherein anupper lid of the petri dish (100) contains the oxygen indicator strip(5)containing resazurin dye which changes color as per the availability of oxygen.
5) The apparatus as claimed in claim 1, wherein the microorganisms comprise anaerobic microorganisms.
6) The apparatus as claimed in claim1, wherein the upper lid of the petri dish (100) contains about 10 gm. of oxygen-scavenging material
7) A method for checking anaerobic status of the petri dish (100)for the growth of anaerobic microorganisms, the method comprising (a) filling an oxygen scavenging absorbent (4) consisting of 10% - 20% of ascorbic acid, 10% - 20% of cobalt chloride, 15% - 40% of sodium bicarbonate, 15% - 40% of ferrous carbonate, 15% - 40% of activated carbon, 0.5% - 4% of magnesium chloride by weight in a transparent polyurethane material which is permeable to gases and has an adhesive backing; (b) preparing an indicator strip(5)of pink-colored resazurin dye; (c) preparing sterilization of the petri dish (100) preferably by using gamma radiation; (d) injecting an upper lid of the petri dish (100) containing the oxygen absorbent (4) with adding 3.0 ml sterile water, by peeling off the seal (7), once the lower (5)lid of the petri dish (100) is inoculated with microorganisms; (e) pressing the lid immediately to create a hermetic seal (7) of the petri dish (100) to exclude ambient air;and (f) observing the change in the pink color of resazurin dye to colorless.
8) The method as claimed in claim 7, the oxygen-scavenging material generates H2 and CO2 when water is added.
9) An oxygen-scavenging absorbent, the absorbent is consisting of 10% - 20% of ascorbic acid, 10% - 20% of cobalt chloride, 15% - 40% of sodium bicarbonate, 15% - 40% of ferrous carbonate, 15% - 40% of activated carbon, 0.5% - 4% of magnesium chloride by weight.

10) The absorbent as claimed in claim 9, wherein the 9.8 grams of absorbent is prepared by mixing 1.24 grams of ascorbic acid, 1.24 grams of cobalt chloride, 2.48 grams of sodium bicarbonate, 2.48 grams of ferrous carbonate, 2.24grams of activated carbon and 0.124 grams of magnesium chloride.