Method and device for inspecting microorganisms of large amount of liquid sample

Abstract

To provide a method and a device which are capable of culturing a large amount of liquid sample on an agar medium and completing culturing.SOLUTION: A method for inspecting microorganism includes providing a device used in microorganism inspection and supporting consumables, where the device sprays polymer dry powder capable of forming hydrogel on an agar medium surface, the polymers are crosslinked to form polymers of a three-dimensional network structure, forming a liquid sample adsorption solidified layer, and at the same time, all microorganisms in the sample are immobilized on the agar medium surface, and microorganisms present in the solidified layer are cultured, and inspected after culturing.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for inoculating large volumes of liquid samples for microbiological testing, and is in the field of microbiological culture technology. [Background technology]

[0002] Traditional microbiological culture testing techniques are classified into agar solid culture and broth liquid culture. These two methods each have their own advantages and disadvantages, and they cannot replace each other; they can only complement each other. Agar plate streaking is the most common method for microbiological testing. A sample is applied or "streaked" onto the surface of a gel plate medium. After incubation, the presence or absence of colonies on the streaked area of ​​the plate is observed to determine microbial growth. The advantage of this method is that colonies can be directly collected for microbial identification and drug susceptibility testing. However, the small sample size required for inoculation makes it difficult to test large liquid samples (such as blood cultures). Broth culture requires a large sample size for inoculation, resulting in a high positive rate, but it does not allow for quantification or isolation / purification of bacteria.

[0003] Microbiological work often requires the microbiological testing of large volumes of liquid samples, such as the microbiological testing of whole blood (or portions) and other body fluid samples in clinical microbiology laboratories, and the microbiological testing of food, drug, water, and object surface samples in food, drug, microbiology, and environmental microbiology laboratories. Therefore, in practical work, a microbiological testing solution that takes into account the advantages of the above two methods is eagerly needed.

[0004] In U.S. Patent No. 4,182,656, Ahnell et al. inoculate a large volume of liquid sample into a vessel containing a culture medium containing a fermentable substrate labeled with carbon-13. During bacterial cultivation, the ratio of carbon-13 to carbon-12 in the gas in the vessel is measured and compared with the value at the start of cultivation, thereby achieving the goal of measuring bacterial growth. U.S. Patents Nos. 4,152,213 and 4,073,691 test for bacteria by monitoring the gas pressure in the vessel. In U.S. Patent No. 5,094,955, Calandra et al. invented a noninvasive method for testing for the presence of microorganisms in clinical samples (e.g., blood or other body fluids) and nonclinical samples. The invention involves introducing a liquid sample into a transparent, sealed vessel containing a culture medium. If microorganisms are present, carbon dioxide is generated by microbial growth, which reacts to a sensor fixed in the vessel. Analysis of receptors attached to the outside of the bottle also provides a non-radioactive, non-invasive means of testing for microorganisms in the presence of interfering substances (e.g., high concentrations of red blood cells).

[0005] Most currently available liquid sample microbial culture devices are based on the testing principle proposed by Calandra et al., but these methods still rely on liquid broth culture. These methods require specialized equipment and consumables, such as sensors and receptors for observing bacterial growth and metabolism. They often require shaking the culture vessel during the incubation process, resulting in high manufacturing costs and a high likelihood of mechanical failure. Furthermore, these devices can only test for the presence of microorganisms; the time it takes to report a positive result correlates with the number of microorganisms in the sample, but they cannot count the bacteria. The biggest problem is that when microorganisms are tested and bacterial identification or bacterial drug susceptibility testing is required, pure colonies must be isolated from the liquid medium using agar solid culture to ensure the reliability of susceptibility testing and bacterial identification. This requires additional time (long testing times can be life-threatening for critically ill patients) and does not meet clinical timeliness requirements.

[0006] The disk method is currently a commonly used solid culture method for large-scale culture of liquid samples. The basic inoculation volume of the liquid sample is about 1-2 ml, which cannot meet the requirements for large-volume specimens such as blood cultures. Furthermore, the culture medium must be prepared in advance for each test experiment, sterilized by autoclave, and a device must be prepared to maintain a constant temperature of 50°C, which is more tedious and difficult to control.

[0007] With the solid plate surface coating method, only very small amounts of liquid sample, about 0.1 ml, can be inoculated, and even on semi-solid media (such as agar), the culture solution cannot swell significantly, so only 5% or less of the initial gel volume can be absorbed.

[0008] Membrane filtration for bacterial testing can capture microorganisms from larger volumes of liquid, but has many drawbacks, including increased manual processing time, high cost, risk of contamination, and difficulty handling samples containing particles.

[0009] In US Patent No. 7,183,073, Hyman et al. optimized and improved a solid culture support to improve the water absorption characteristics of the solid medium for the purpose of inoculating large amounts of liquid samples. This culture device includes a polymeric immobilizing layer with voids between the polymer gel matrix. The fluid sample is applied to the immobilizing layer, and the fluid is absorbed by the immobilizing layer, retaining the microorganisms on its surface. Although the amount of liquid absorbed is said to be approximately 2.5–5 mL, the entire absorption process takes more than 10–20 hours. Due to the slow absorption rate, the solid plate cannot be turned over, making it susceptible to contamination by environmental microorganisms during the culture process. At the same time, the polymeric immobilizing layer swells and deforms significantly after absorbing a large amount of liquid, destroying the solid gel structure and affecting microbial testing. Furthermore, the ratio of nutrients necessary for microbial growth becomes unstable due to changes in the culture medium, affecting microbial testing. At the same time, conventional solid agar medium systems cannot be used. The manufacturing process is complex, requiring high pressure, vacuum, and UV irradiation to initiate polymerization. Summary of the Invention [Problem to be solved by the invention]

[0010] In this method, a large volume of liquid sample containing microorganisms is injected or dripped onto the surface of an agar medium using a sterile method. The liquid in the sample is quickly absorbed by the sprayed dry polymer powder, and the polymer molecules crosslink through their side chains to form a stable gel. The liquid sample quickly forms a compact hydrogel-like liquid sample adsorption and solidification layer on the solid medium of the agar plate, and the microorganisms in the sample are retained in the solidified layer on the surface of the agar medium. After the agar plate is inverted and bacterial cultured using conventional solutions, individual microbial colonies can be easily isolated and cultured on solid medium for further microbiological testing.

[0011] The device of the present invention comprises a dry powder sprayer, a liquid-solidifying dry powder consumable (various polymer materials) packaged in a sealed container, and a sample container for auxiliary use.

[0012] The dry powder sprayer consists of a computer-controlled system, a Petri dish mechanical transport system, a dry powder loading and metering device, and a dry powder mixing and spraying system. The dry powder spraying process involves the following steps: S1: Inoculate a large amount of liquid sample onto an agar medium (aseptic method by quantitative injection or dropwise addition); S2: Place the agar Petri dish on the device, automatically transport it inside the device, and open the Petri dish; S3: Select a predetermined amount of sterile polymer dry powder to spray onto the agar medium based on the liquid volume; S4: Spray using the dry powder mixing and spraying device, and the polymer crosslinks to form a three-dimensional mesh-like adsorption layer; S5: Fix microorganisms on the sample surface; S6: Fix the microorganisms in the sample to the agar medium surface after spraying; S6: After spraying, the Petri dish is automatically removed from the device, and the inside of the device is covered with the Petri dish during transport, and the Petri dish is automatically removed from the device; S7: Cultivate the microorganisms in the solidified layer, forming colonies for testing.

[0013] The present invention allows for sample manipulation of different liquid volumes from 0.5 ml to 10 ml. Sterile procedure is guaranteed throughout the procedure. Reagent containers are sterile and airtight, and the dry powder piping in the device is airtight.

[0014] The polymers used in the present invention that can form hydrogels may be natural or artificial materials, but all share common physical properties, including the following: 1) They are capable of rapidly absorbing fluids from aqueous solutions or suspensions, and the polymers swell but do not dissolve. 2) They are capable of forming gels or highly viscous solutions, preventing the overall movement of fluids and meeting the requirements for microbial testing. 3) Chain polymers form three-dimensional networks by blending different polymers or by adding crosslinkers, and form colloids at room temperature. 4) They do not affect the growth of microorganisms.

[0015] The polymers used in this invention have sufficient water absorption capacity to absorb large amounts of liquid from samples. By mixing or adding an appropriate crosslinking agent to form a three-dimensional polymer colloid, not only is water absorption guaranteed, but colloidal performance and liquid absorption speed are also guaranteed, preventing sample flow and dripping. Liquid absorption is usually completed within a few minutes to an hour. For routine culture, the Petri dish can be turned over in a short time, ensuring that the formed colonies do not experience flow or diffusion problems.

[0016] The sample container of the present invention is a sterile, leak-proof, airtight container. To enhance the effectiveness of microbial testing, one or more of the following may be added to the support sample container: nutrients necessary for microbial growth, microbial growth regulators, hemolytic agents, dissolving agents, antibiotic neutralizers, and selective bacterial growth agents. The bulk fluid sample of the present invention may be an aqueous liquid such as whole blood (or a portion thereof), other bodily fluids, manufacturing fluids, food samples, etc. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a schematic diagram of the principle of microbiological testing of a large volume of liquid sample. [Figure 2] FIG. 1 is a schematic diagram of a dry powder atomizer. [Figure 3] Figure 10 shows the bacterial culture results after a large volume of liquid sample was adsorbed with a dual gum blend dry powder to form a solidified layer. [Figure 4] FIG. 10 shows the results of forming adsorbed and solidified layers of multiple blood samples using multi-gel mixed dry powder scheme 2. [Figure 5] FIG. 1 shows the growth of Staphylococcus aureus in blood samples 48 hours before and after treatment with adsorbed antibiotics. DETAILED DESCRIPTION OF THE INVENTION

[0018] Figure 1 is a schematic diagram illustrating the principle of this method. Reference numeral 80 denotes a conventional agar medium, but this method is not limited to any particular medium and can be used with any agar medium commonly used in microbiology. The air source 10 uses airflow control to spray a dried polymer powder onto a Petri dish inoculated with a large amount of liquid sample. These polymers must be able to rapidly adsorb the liquid in the sample and, at room temperature, transform into a hydrocolloid, forming a three-dimensional structure, forming a strong solidified layer on top of the sample, which aids in sample immobilization / adsorption. By forming a three-dimensional hydrocolloid structure at room temperature and forming a strong solidified layer on top of the agar medium, the solidified layer's properties aid in sample immobilization / adsorption, allowing microorganisms to grow locally and form colonies, similar to traditional agar culture, improving microbial colony testing capabilities. In the figure, 10, 20, 30, 40, 50, 60, 70, and 80 represent the air source, mixing chamber, polymer, air pressure, spray-dried powder, Petri dish, liquid sample, and agar medium, respectively.

[0019] 2 is a schematic diagram of a dry powder sprayer. The dry powder sprayer is composed of a computer control system (not shown), a Petri dish mechanical transport system 100, a dry powder loading and metering device 200, and a dry powder mixing and spraying system 300.

[0020] The dry powder container must be airtight, moisture-proof, and sterile. The dry powder container of the dry powder loading and metering device 200 can be connected to a container containing dry powder reagent via an interface, but this connection must be airtight. The reagent container containing the dry powder reagent is designed as a sealed, sterile package with an aluminum-plastic sealing film on one end. After the device is connected, a film punching device pierces the aluminum-plastic sealing film and connects it to the dry powder container. After the dry powder reagent is consumed, the reagent container can be directly replaced with a clip for single use.

[0021] The dry powder loading and dispensing device (quantity adjusting device) of the dry powder dispensing device 200 is designed with a dry powder dispensing hole, and the size of the hole is used to measure the amount of dry powder reagent from the dry powder loading container to the reagent tank and transport it to the dry powder injection tank of the dry powder mixing and spraying system 300. The dispensing device is designed with a volume hole or multiple identical volume holes, and the number of times the dispenser is loaded can be controlled to add various amounts of dry powder. That is, a predetermined amount of dry powder can be added using a single hole capacity corresponding to any number of dry powder amounts. The amount of dry powder can also be increased by any multiple of the single hole capacity. The weight of the dry powder allows the dry powder to fall naturally from the dry powder dispensing container into the dry powder dispensing hole. Then, a plate containing agar medium is transferred to the dry powder mixing and spraying system 300 by a Petri dish mechanical transport system, and the dry powder is sprayed onto the plate surface using a gas mixture flow. The dry powder is then uniformly dispersed over the plate surface by the air jets of the dry powder mixing and spraying system 300.

[0022] The entire process is carried out in a sealed space, and the Petri dish is automatically transported into the device by the Petri dish mechanical transport system 100. At the same time, the Petri dish is opened inside the device, and after the dry powder is sprayed, the inside of the device is covered with the Petri dish, and the Petri dish is automatically transported outside the device. In this way, contamination can be prevented.

[0023] <Selection of highly absorbent polymer> The superabsorbent material is a hydrophilic high molecular weight polymer material. Common superabsorbent polymers are classified into natural and modified polymer-based superabsorbents and synthetic absorbent materials.

[0024] Natural and modified polymer-based superabsorbents are obtained from plants, animals, or microorganisms by physical or physicochemical methods. They are classified into the following five types based on the origin of the raw materials:

[0025] Plant polysaccharide gums are thickening biopolymers derived from plant roots, stems, leaves, seeds, and fruits. Commonly used plant gums include blueberry gum, konjac gum, locust bean gum, carrageenan, guar gum, tamarind gum, and gum arabic. Animal protein polysaccharide gums are derived from animal skin, bones, tendons, and milk. They have thickening properties and are primarily composed of protein biopolymers such as chitosan, gelatin, casein, whey protein concentrate, and fish gelatin. Microbial polysaccharide gums are derived from microbial metabolites, such as xanthan gum, gellan gum, and yeast polysaccharides, which have thickening properties for biopolymers. Algal polysaccharide gums are extracted from seaweed and can thicken biopolymers such as agar, alginic acid (salt), propylene glycol alginate, red algae gum, and brown algae fucoidan.

[0026] Chemically modified gums are new gums such as methylcellulose, modified starch, soluble starch, hydroxymethylcellulose, and modified chitosan, which are based on the above natural gums but have their structure altered and obtained through processes such as chemical synthesis.

[0027] Common natural superabsorbent materials include natural starch, vegetable gums, animal gums (casein), pectin, chitin, and water-soluble alginic acid derivatives. Examples include guar bean gum, acacia bean gum, tara gum, acacia gum, tamarind gum, karaya gum, gum arabic, ghatti gum, yarrow gum, carob gum, succinic acid polysaccharides, xanthan gum, gellan gum, and reduced-sugar glue. Other natural materials include alginic acid, chitosan, agar, agarose, soy protein, gluten, and sericin.

[0028] Semi-synthetic modified superabsorbents are chemically modified versions of the natural materials listed above. Starch-based materials include sodium carboxymethyl starch, starch-grafted acrylate hydrolysate, starch-grafted acrylate polymer, starch-grafted acrylamide polymer, starch-grafted styrene sulfonate polymer, and starch-grafted xanthate acrylate. Cellulose-based materials include carboxymethyl cellulose, cellulose-grafted acrylate polymer, cellulose-grafted sulfonate acrylate, cellulose-grafted acrylamide polymer, and cellulose-grafted acrylonitrile hydrolysate. Guar gum-based materials include hydroxyethyl guar gum, hydroxypropyl guar gum, carboxymethyl hydroxyethyl guar gum, and carboxymethyl hydroxypropyl guar gum.

[0029] Synthetic water-absorbing materials include: Polyvinylates: crosslinked polyacrylates, polyacrylamides, hydrolyzed acrylate-vinyl acetate copolymers, copolymers of acrylic acid and acrylamide, sodium acrylate-vinyl alcohol copolymers (copolymers of methyl acrylate and vinyl acetate), hydrolyzed polyacrylonitrile, hydrolyzed poly(methyl acrylate), hydroxypropylene methacrylate, crosslinked polyacrylates, polyacrylamides, hydrolyzed acrylate-vinyl acetate copolymers, copolymers of acrylic acid and acrylamide, sodium acrylate-vinyl alcohol copolymers (copolymers of methyl acrylate and vinyl acetate), hydrolyzed polyacrylonitrile, and polyhydrogen methacrylates. Polyvinyl alcohol: crosslinked polyvinyl alcohol-maleic anhydride copolymers, polyvinyl alcohol-acrylic acid graft copolymers, vinyl acetate-vinyl acrylate cohydrolyzates, vinyl acetate-maleic anhydride copolymers, crosslinked polyvinyl alcohol, and elastomers obtained after freezing and thawing polyvinyl alcohol. Polyoxyethylene: polyethers.

[0030] All superabsorbent polymers have thickening properties, but only a few have the ability to form gels. Their aqueous solutions can form gels, such as polysaccharides like agar, carrageenan, and gelatin. Most superabsorbent polymers, such as tamarind gum, locust bean gum, konjac gum, xanthan gum, and guar gum, cannot naturally form gels.

[0031] Gelling refers to a special dispersion system formed when, under certain conditions, colloidal particles or polymers in a sol or solution bond with each other to form a spatial network structure (three-dimensional network structure) that prevents the flow of the system, and the voids in this structure are filled with a liquid as a dispersion medium.

[0032] Hydrogels are three-dimensional polymer networks containing large amounts of water. The presence of hydrophilic functional groups, such as -OH, -CONH2, -CONH, and -SO3H, allows the polymers in the network to bind large amounts of water. As a result, hydrogels can remain swollen in water without dissolving. There are various types of hydrogels, which can be classified according to different criteria. Depending on the type of crosslinking, hydrogels can be classified as physically crosslinked, chemically crosslinked, or a mixture of the two. Physical hydrogels are generally formed by physical interactions such as ions, hydrogen bonds, chain entanglement, and other interaction forces. Chemical gels are three-dimensional polymer networks formed by covalent crosslinking and are more stable than physical gels. They are also known as permanent gels. Chemical gels are generally formed by the polymerization of monomers and crosslinkers that initiate hydrogel polymerization.

[0033] Conventional methods for preparing hydrogels mainly include: (i) reactive polymerization initiated by free radicals generated by ionizing radiation, (ii) the formation of intertwined polymers through chemical reactions, and (iii) physical interactions such as entanglement, electrostatics, and microcrystal formation.

[0034] Physical crosslinking methods for preparing hydrogels involve interpolymer physical forces, such as electrostatic interactions, hydrophobic interactions, hydrogen bonding, and interchain entanglement. These temporary physical crosslinks can form three-dimensional networks of hydrogel polymers. These forces allow polymers to interact with multivalent ions, polyelectrolytes, and hydrophobic groups to form physical gels. While this preparation method is relatively simple and easy to operate, the resulting gels have poor mechanical properties, are easily damaged, and may gradually degrade under external conditions. The gelation process is generally reversible. A typical physical gel is polyvinyl alcohol hydrogel, which is formed by repeatedly freezing and thawing an aqueous solution of polyvinyl alcohol. It can be decrosslinked and reconstituted under heating. Agarose dissolves in water at high temperatures and forms a solution. Upon cooling, it crosslinks into a network structure, forming a gel. Radiation crosslinking is a method in which certain polymers undergo spontaneous crosslinking via radiation-induced free radicals upon exposure to high-energy radiation (e.g., gamma rays or electron beams). Sodium alginate can be cross-linked by Ca2+ to form a hydrogel, and gelatin and agarose can also be cross-linked in the presence of hydrogen bonds.

[0035] Chemical gels are hydrogels formed by covalent cross-linking of molecules, and synthetic gels are generally of this type. These covalent cross-links are typically formed by copolymerization of monomers and cross-linking agents, or by the interaction of functional groups in the molecular chains. Covalent cross-links are strong, and the gelation process is generally irreversible.

[0036] The crosslinking method required by the present invention must satisfy the following requirements: it must be able to produce a polymer at room temperature in a short time without additional processes such as heating, high pressure, or irradiation, and it must ensure both high water absorption and the formation of a stable three-dimensional network structure that forms a gel.

[0037] Highly water-absorbent polymers are long-chain polymers with little network crosslinking. When these materials crosslink into a network structure and form a gel, their water absorption capacity decreases and water precipitates. Therefore, controlling the degree of crosslinking is very important, and the gel formed by the polymer must maintain cohesive strength or high viscosity under the conditions of use in order to maintain water absorption and crosslink to form a gel. The gel must maintain its integrity even during changes in volume and temperature required for its use. It is also desirable that the polymer is not easily degraded by cultured microorganisms. Furthermore, the interchain crosslinking or entanglement of the gelling polymer must be high enough to maintain gelation or high viscosity, yet low enough to allow a high degree of swelling. More specifically, regardless of the material used (natural, synthetic, semi-synthetic, or other), it is preferable to provide a network of interconnected polymer chains that is flexible enough to absorb liquid without disrupting the network.

[0038] Common crosslinkers include transition metal crosslinkers such as aluminum, chromium, titanium, zirconium or Group IV metal compounds; organic transition metal crosslinkers such as organozirconium crosslinkers and organotitanium crosslinkers; boron crosslinkers such as borax, boric acid and organoboron crosslinkers; and chemical crosslinkers such as glutaraldehyde crosslinkers, formaldehyde, N,N'-methylenebisacrylamide (MBA) crosslinkers, genipin, and ethylene glycol diglycidyl ether.

[0039] <Double gel blend gel> A double gel blend gel refers to a gel formed by blending (combining) two absorbent polymer polysaccharides.

[0040] At room temperature, there are colloids (water-absorbent polymers) that do not form gels by themselves; these are called non-gelling polysaccharides. However, non-gelling polysaccharides form gels when combined with other polysaccharide gels.

[0041] Xanthan gum also has a high viscosity at low concentrations and is a pseudoplastic fluid, but it cannot form elastic gels. Xanthan gum exhibits significant synergistic effects with many non-gelling polysaccharides, resulting in mixed gels with viscosities several times higher than those of the same concentration of a single gel, or even a jelly-like gel. This phenomenon is known as synergistic thickening and gelation. This is primarily due to the double helix structure of xanthan gum molecules, which easily form chimerizations with polysaccharide molecules containing β-1,4 linkages. This interaction is exploited to maximize gel strength. The coexistence of two polysaccharide polymers in the appropriate ratio maximizes the synergistic effect of the two polysaccharide molecules, resulting in the strongest gelling ability and maximum gel strength.

[0042] Similarly, konjac glucomannan (konjac gum) has a high apparent viscosity and does not exhibit gel properties in non-alkaline solutions. However, when xanthan gum is added to a konjac glucomannan solution, the apparent viscosity of the konjac glucomannan-xanthan gum composite gel gradually increases with increasing xanthan gum content, and then gradually decreases after the mass ratio of konjac glucomannan to xanthan gum reaches a maximum. This indicates that when konjac glucomannan and xanthan gum interact at a certain ratio, they not only thicken but also possess gelling properties. The powerful synergistic effect of konjac glucomannan and xanthan gum is as follows: konjac glucomannan molecules form a three-dimensional network structure in the smooth part of the molecule, without the xanthan gum double helix structure in the branched chains and secondary bond connections. When the composite gel concentration reaches a certain value, an elastic gel is formed, and the gel strength increases with increasing gel concentration. Furthermore, when the mixing ratio of xanthan gum and konjac glucomannan was 70:30 and the total polysaccharide concentration was 1%, the maximum synergistic effect was reached and a solid gel could be formed.

[0043] <Multi-gel mixed gel> Multi-gel mixtures refer to the mixture of two or more gels with different properties due to their different chemical compositions and structures. When these different substances coexist, their properties often change to varying degrees due to the interactions and influences between them. Compared with monomeric gels, mixed gels have obvious advantages in gel formation.

[0044] By mixing various gums, various complementary effects of each gum can be exhibited, which can expand the range of use of the gum and improve its performance. This synergistic effect can improve the rheological properties of the colloid, control the colloid formation rate, and improve the uniform and transparent appearance of the colloid.

[0045] There is a clear gel synergy between locust bean gum, xanthan gum, guar gum, and gellan gum.

[0046] Locust bean gum itself does not have gelling properties, but it has good gelling synergy with other hydrophilic colloids.

[0047] Guar gum is a water-soluble polymer with extremely high water absorption. Its structure is a curled globular structure containing numerous hydrophilic hydroxyl groups, most of which are located on the interior of the molecule, allowing for intramolecular self-crosslinking through intermolecular forces. Guar gum and modified guar gum can be mixed with linear polysaccharides such as xanthan gum, agar gum, K-type carrageenan, locust bean gum, sodium alginate, konjac gum, gellan gum, and starch to form colloids.

[0048] <Stability of mixed colloids> The stability of hydrogels formed by different types of gums varies, and acidic and alkaline conditions have a significant effect on some gels. When bacteria grow, the pH changes, dissolving the formed hydrogels. Various enzymes produced by bacteria also dissolve the formed hydrogels.

[0049] Xanthan gum, konjac gum, gellan gum, guar gum, hydroxypropyl guar gum, cationized guar gum, xanthan gum-konjac gum composite gum, and Yellow Magic Acacia Melon composite gum all have excellent acid, alkali, and enzyme stability. They are highly stable in both acidic and alkaline environments and can be used under both acidic and alkaline conditions. Their solution viscosity remains relatively stable in the pH range of 2 to 12. Conventional microbial and industrial enzymes, such as proteases, cellulases, pectinases, and amylases, have no effect on these gums.

[0050] <Application of crosslinking agents> To ensure the colloidal properties of the solidified liquid layer and enable the formation of a stable three-dimensional network of water-absorbing polymers, the present invention uses a chemical cross-linking agent for bacterial testing in large volume liquid samples.

[0051] <Addition of nutrients, regulators, hemolytic agents, dissolving agents, antibiotic neutralizers, and selective bacterial growth agents> To improve the effectiveness of microbiological testing, one or more of the following substances can be added to the test system: nutrients necessary for microbial growth, microbial growth regulators, hemolytic agents, lytic agents, antibiotic neutralizers, and selective bacterial growth agents. This design allows these substances to be pre-added to the appropriate container depending on the specific test purpose. For example, antibiotic neutralizers and anticoagulants can be added to blood culture tubes to resolve specimens that interfere with the removal of cellular immune factors such as WBCs, humoral immune factors such as antibodies and complements, and bacterial growth factors such as antibiotics and other drugs. These containers can also be designed for specific purposes, such as neutralizing disinfectants for environmental monitoring, drug testing, and food testing.

[0052] The hemolytic or solubilizing agent dissolves or dissolves blood cells or other human tissue substances in the specimen and promotes water absorption. Commonly used hemolytic agents include saponin, mauve saponin, Tween, monolauric acid glyceride or other surfactants, and the solubilizing agent may be a protease.

[0053] The microbial growth regulator may be various growth factors for bacteria, such as tryptone, soy peptone, peptone, malt extract, glucose, hemoglobin chloride, cystine, disulfides, menaquinone, coenzyme I.

[0054] Antibiotic neutralizers can eliminate the effects of antibiotics in a specimen on bacterial culture, improving and / or speeding up the culture of microorganisms in a specimen. Examples of such neutralizers include various resins, gums, carbon-based substances (e.g., activated charcoal), and antibiotic-degrading enzymes (e.g., β-lactamase).

[0055] Selective growth agents for bacteria added during selective culture: for example, lactose, bile salts, K2HPO4, MgSO4, magnesium sulfate, disulfides, and SDS can be added to E. coli; glucose, yeast extract, NHSiCl4, citric acid, or tartaric acid can be added to fungi and other acid-tolerant microorganisms.

[0056] <How to sterilize consumables> To ensure the sterility of disposable consumables provided to users, such as absorbable dry powder reagents and liquid crosslinkers, consumables that are not suitable for autoclave sterilization can be sterilized by radiation. Radiation sterilization does not affect the appearance or physical properties of various adhesive powders, and liquid crosslinkers remain unchanged in appearance and physical properties and are colorless, transparent aqueous solutions. Conventional 8K irradiation does not achieve sterility. To ensure sterilization, 25K irradiation is required at least twice a day. Consumables can also contain one or more of the following nutrients necessary for microbial growth: microbial growth regulators, hemolytic agents, lytic agents, antibiotic neutralizers, or bacterial selective growth agents, and can be packaged in sample containers.

[0057] <Example 1> Xanthan gum and konjac glucomannan dry powder Dry powder was prepared by mixing xanthan gum dry powder and konjac dextran dry powder in a ratio of 70:30, and after thorough mixing, it was placed in a sealed container, autoclaved at 121°C, and dried overnight at 80°C for use.

[0058] <Method> A liquid sample was dropped onto an agar medium Petri dish, and under sterile conditions, xanthan gum konjac glucomannan dry powder was sprayed uniformly at a 4% w / v concentration under high pressure onto the Petri dish containing a large amount of liquid sample. These polymers rapidly adsorbed the liquid in the sample and formed a three-dimensional hydrogel structure at room temperature, forming a strong solidified layer on top of the agar medium, which helped to fix and absorb the sample. The dish quickly absorbed and dried saline solution. After 1-10 minutes, the Petri dish was inverted and placed in a 37°C incubator. Microorganisms then grew locally, forming colonies, similar to conventional agar medium culture. The condition of the medium and bacterial growth could be continuously monitored for 1-14 days. For observation at a 4% ratio, 0.04g of the above dry powder was sprayed evenly onto 1ml of saline. Results: After overnight incubation, all of the liquid was absorbed into the gel, and isolated bacterial colonies were easily observed on the gel surface and could be directly collected. The results are shown in Figure 3. Figure 3 shows the bacterial culture results after a large amount of liquid sample was absorbed by the double-adhesive composite dry powder and a solidified layer was formed. Dual Gum Compound Fixer Dry Powder: Xanthan Gum Dry Powder: Konjac Glucomannan Glucan Dry Powder = 70:30 Medium type: Nutrient agar dish Liquid sample: 3 ml of non-sterile saline solution.

[0059] Example 2: Multi-gel mixed dry powder scheme 1 (Yellow Magic Acacia Melon 6211 mixed dry powder) Based on the optimized formulation of the two-component dry powder solidifier, we found an optimal formula for a multi-component dry powder solidifier. This improved water absorption allows the multi-gel mixed dry powder to absorb more than 10ml of saline, ensuring excellent colloidal performance. Initial absorption is more rapid, with 1-3ml of saline being absorbed in 1-5 minutes. For volumes of 4ml or more, the liquid can be drained and the dish inverted for 10-30 minutes before incubation. This resolves the issue of hydrogel stability, resulting in a stronger gel the next day and for long periods (1-14 days). The hydrogel's properties remain stable and unaffected by bacterial growth, pH changes, and the production of various enzyme colloids.

[0060] After preliminary screening of mixtures including xanthan gum, guar gum, hydroxypropyl guar gum, konjac gum, gellan gum, locust bean gum, sodium carboxymethylcellulose, sodium carboxymethyl starch, and sodium polyacrylate, the following four gums were selected for blending testing. Xanthan gum: Does not form colloids on its own, has uniform, fine particles, high absorbency, viscosity, and low cost. Konjac gum: Can form colloids on its own, has coarse particles, high water absorption, slow water absorption, and uneven distribution of dry powder. Locust bean gum: Cannot form colloids on its own, has very dilute solutions and good fluidity. It aids in uniform dispersion of dry powder and provides excellent synergistic effects of side-branch crosslinking. Hydroxypropyl guar gum: Cannot form colloids on its own, has excellent water absorption, and can absorb water quickly. After optimizing multiple blend combinations, the blend ratio was determined to be 6:2:1:1.

[0061] <Multi-component dry powder solidification formulation> Xanthan gum: konjac gum: locust bean gum: hydroxypropyl guar gum = 6:2:1:1:1 were thoroughly mixed, placed in a sealed container, sterilized in an autoclave at 121°C, and dried overnight at 80°C before use.

[0062] <Method> Different amounts of liquid samples were poured or dropped into agar medium dishes, and the above dry powder was sprayed at a 3% w / v ratio under sterile conditions. The water absorption in the dishes was observed, and once the liquid had been absorbed and dried, the dishes were placed in a 37°C incubator and the condition of the culture medium and bacterial growth were continuously observed for 1 to 14 days.

[0063] The firmness of the bond between the solidified layer and the agar medium, the absorption of the sample by the solidified layer, and the growth and colony formation of microorganisms were observed.

[0064] Example 3: Multi-gel mixed dry powder scheme 2 (Junction Hydroxylan 721 mixed dry powder) Unlike saline, blood contains a large amount of proteins, inorganic salts, and various other substances. The presence of these substances affects the formation of the three-dimensional structure of the hydrogel and the stability of its colloidal properties. Therefore, we established a multi-gel mixed dry powder scheme 2 for the blood adsorption and solidification scheme.

[0065] <Multi-component dry powder solidification formulation> Gellan gum: hydroxypropyl guar gum: cationized guar gum = 7:2:1 were thoroughly mixed, placed in a sealed container, sterilized in an autoclave at 121°C, dried overnight at 80°C, and prepared for use.

[0066] <Method> Using the same methods as in Example 2, the firmness of the bond between the solidified layer and the agar medium, the absorption of the sample by the solidified layer, and the growth and colony formation of microorganisms were observed.

[0067] The results are shown in Figure 4. Figure 4 shows the results of adsorbing multiple blood samples and forming a solidified layer using Multi-Gel Mixed Dry Powder Scheme 2. Mixed solidifying agent: dry powder gellan gum: hydroxypropyl guar gum: cationized guar gum = 7:2:1 Media type: Columbia blood agar plates Liquid sample: 10 ml normal human anticoagulated blood.

[0068] Example 4: Solidification of a large volume of liquid sample In the present invention, in order to absorb a large amount of liquid in the sample, ensure the colloidal properties of the solidified liquid layer, and form a stable three-dimensional network structure in the water-absorbing polymer, a large amount of liquid sample (20 ml) was solidified by adding a chemical crosslinking agent to a single colloid, hydroxypropyl guar gum, and a multi-component blend gum. Ingredients: Hydroxypropyl guar gum, Yellow Magic Acacia Melon 6211 (homemade) Crosslinker: Organic zirconium crosslinker (DuPont Tyzor 212) Sample types: Saline and anticoagulated blood

[0069] <Method> A dry powder mixture of hydroxypropyl guar gum and Yellow Magic Acacia Melon 6211 was sterilized in an autoclave and prepared for use. When sterilizing by autoclave, it must be sealed and moisture-proof. The dry powder was sprayed onto a blood plate agar medium containing 20 ml of anticoagulated blood sample (or saline) and an organic zirconium crosslinker. After leaving the medium at 37°C for 1 hour, the medium was turned upside down and placed in a 37°C incubator for observation for 3-5 days.

[0070] The results are shown in Table 1. Table 1. Colloid formation in the solidified layer of a 20 ml saline sample JPEG2025177930000002.jpg65170Table 2 Colloid formation in the solidified layer of a 20 ml anticoagulated blood sample JPEG2025177930000003.jpg53170

[0071] Example 5: Effect of organozirconium crosslinking agents on bacterial growth The effects of organozirconium crosslinkers on bacterial growth were observed, and a safe amount of crosslinker to be used was selected to determine whether organozirconium crosslinkers could be used in bacterial testing.

[0072] JPEG2025177930000004.jpg4170Culture bottle (30ml pediatric bottle), 3. Clinical isolates: Freshly prepared bacterial liquid for clinical drug susceptibility testing.

[0073] <Method> First, a bacterial solution was prepared with saline and tested using a turbidity meter to obtain a bacterial solution with a concentration of 0.5 mcg (10 cfu / ml). The bacterial solution was then diluted to a final concentration of 3 cfu / ml. Two bioMérieux blood culture bottles (manufactured by France) were used as the measurement and control tubes, respectively. The above bacterial solution was then injected into each of the two bioMérieux blood culture bottles (manufactured by France), with an inoculation volume of 1 ml per bottle. 0.9 ml of 10% organic zirconium crosslinker (0.3% (v / v) in the system) was added to the measurement bottle, and the incubation was completed within 5 minutes. JPEG2025177930000005.jpg10170

[0074] The results are shown in Table 3. Table 3. Effect of organozirconium crosslinkers on bacterial growth JPEG2025177930000006.jpg132170

[0075] The results show that the system containing 0.3% (v / v) organozirconium crosslinker does not affect bacterial growth and is sufficient for use in routine microbiological testing procedures.

[0076] Example 6: Resin blood collection tubes improve bacterial testing of clinical blood specimens Clinical patients often use large amounts of antibiotics for long periods of time before blood cultures are performed, which seriously affects the positive rate of bacterial isolation cultures in blood. Antibiotic neutralizers can eliminate the effects of antibiotics in specimens on bacterial culture, improving and / or speeding up the culture of microorganisms in test specimens.

[0077] Measurement tube: 8 ml blood collection tube containing 0.6 g of macroporous adsorption resin and SPS anticoagulant, control tube: 8 ml blood collection tube containing only SPS anticoagulant.

[0078] Preparation of mock-positive blood samples: The final bacterial concentration of the mock samples was adjusted to 1–10 cfu / ml using saline supplemented with Staphylococcus aureus ATCC25923.

[0079] Four milliliters of blood was collected from patients undergoing clinical antibiotic treatment, separated, and injected into a measurement tube and a control tube, 2 ml each.

[0080] The Yellow Magic Acacia Melon 6211 mixed dry powder was sterilized in an autoclave and prepared for use. When sterilizing by autoclave, it must be sealed and moisture-proof. 2 ml of the above anticoagulant sample was sprayed over a blood plate agar medium and left to stand at 37°C for 1 hour. The medium was then turned upside down and placed in a 37°C incubator for incubation.

[0081] The results are shown in Figure 5. Figure 5 shows the growth of Staphylococcus aureus in blood samples 48 hours before and after antibiotic adsorption treatment. Mixed hardener dry powder xanthan gum: konjac glucomannan gum: acacia bean gum: hydroxypropyl guar gum = 6:2:1:1 Culture medium type: Columbia blood agar dish Liquid sample: 2 ml anticoagulated blood Strain: Staphylococcus aureus ATCC25923. The upper graph A shows 2 ml of blood from a patient who had been treated with antibiotics and had been treated with adsorption resin, while the lower graph B shows 2 ml of blood from a patient who had been treated with antibiotics without treatment with adsorption resin. For colonies of Staphylococcus aureus ATCC25923 cultured for 48 hours, the results from the measurement tube were significantly better than those from the control tube, indicating that the adsorption resin has a significant effect on removing antibiotics and has a good application effect.

[0082] The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0083] Compared with the prior art, the present invention has the following advantages:

[0084] 1. The method for testing microorganisms in large volumes of liquid samples of this invention relies on the traditional solution of solid culture, by directly solidifying a large volume of liquid sample, integrating the sample with agar medium, and completing the culture of the large volume of liquid sample on solid agar medium. This method overcomes the traditional need to first culture a large volume of liquid sample in broth and then culture it on solid agar medium for solid isolation, simplifying the culture procedure and shortening the culture and testing time. This method integrates liquid culture and solid culture, integrates bacterial culture and bacterial isolation, quantifies microorganisms, and improves the positive rate of microbial culture.

[0085] 2. This invention discloses a method and apparatus for isolating and culturing microorganisms from large volumes of liquid samples. This method uses conventional agar media. A fixed amount of the liquid sample to be tested is aseptically poured or dripped onto the surface of the agar media. A sterile polymer dry powder is then sprayed onto the agar media using a dry powder sprayer specially designed for this invention. The polymer crosslinks to form a three-dimensional mesh-like adsorption layer, rapidly immobilizing the microorganisms from the sample. The microorganisms in the sample are quickly immobilized on the agar media surface, and the microorganisms in the solidified layer are cultured and grown to form colonies for testing, which can then be counted. These colonies can also be directly used for further processing, such as bacterial identification, drug susceptibility testing, or other microbiological analysis and testing.

[0086] 3. The microorganism testing method of the present invention involves spraying a dry powder of a highly absorbent material onto a liquid sample, absorbing a large amount of liquid, while simultaneously using a polymer to form a three-dimensional network structure and form a gel at room temperature. This allows for excellent colloidal transparency, controllable properties, and does not affect microbial growth. The present invention has a fast water absorption rate, completing the absorption process within minutes to hours, making it easy to cultivate isolated bacterial colonies. The pure colonies can then be directly used for bacterial identification, drug susceptibility testing, and other related procedures.

[0087] 4. The microorganism testing method of the present invention has a high water absorption capacity. A 90 mm diameter plate medium can absorb up to 20 mL of liquid, while a regular agar medium can only absorb 0.2 mL of liquid in 30 minutes.

[0088] 5. The method for microbial testing of large volumes of liquid samples according to the present invention has a short incubation time, typically within 3 days, with most cases completed within 18 hours. In current clinical practice, the incubation period for fluids such as blood, pleural fluid, ascites, synovial fluid, and pericardial fluid is typically 5-7 days.

[0089] 6. The method of the present invention significantly expands the scope of application of the solid agar plate culture method. Currently, in microbiological testing in medicine, industry, food, etc., liquid broth culture methods are generally used for large amounts of liquid samples, but the present invention proposes a new program.

Claims

1. A method for testing microorganisms, comprising providing an apparatus for use in microorganism testing and supporting consumables, wherein the apparatus sprays a hydrogel-forming polymer dry powder onto the surface of an agar medium, and these polymers are cross-linked to form a three-dimensional network structured polymer, creating a liquid sample adsorption solidification layer, and simultaneously all microorganisms in the sample are immobilized on the surface of the agar medium, and the microorganisms present in the solidification layer are cultured and then tested.

2. 10. An apparatus for use in the method of claim 1, wherein the apparatus is a dry powder sprayer comprising a computer control system, a Petri dish mechanical transport system, a dry powder loading and measuring device, and a dry powder mixing and spraying system, wherein the measuring device of the dry powder loading and measuring device is designed with a dry powder measuring hole, and the dry powder falls naturally from the dry powder measuring container of the dry powder loading and measuring device into the dry powder measuring hole using the weight of the dry powder, the Petri dish mechanical transport system moves the plate containing the agar medium to the dry powder mixing and spraying system, and the dry powder is sprayed onto the surface of the plate using a gas mixed flow.

3. 2. A liquid sample adsorption / solidification layer for use in a microorganism test, which is used in the method according to claim 1, wherein the liquid sample adsorption / solidification layer is made of a water-absorbent polymer material, and includes a natural, semi-synthetic modified, or synthetic highly water-absorbent material; The natural superabsorbent material is natural starch, vegetable gum, animal gum, pectin, chitin, seaweed derivatives, guar gum, konjac gum, locust bean gum, tamarind gum, gum arabic, carrageenan, xanthan gum, gellan gum, alginic acid (salt), chitosan, agar, agarose, gelatin, casein, or casein; the semi-synthetic modified superabsorbent material is sodium carboxymethyl starch, starch-grafted acrylate polymer, starch-grafted acrylamide polymer, carboxymethyl cellulose, cellulose-grafted acrylate polymer, cellulose-grafted acrylamide polymer, hydroxyethyl guar gum, hydroxypropyl guar gum, carboxymethyl hydroxyethyl guar gum, or carboxymethyl hydroxypropyl guar gum; The synthetic highly water-absorbent material is a liquid sample adsorption / solidification layer for microorganism testing, which is a cross-linked polyacrylate, polyacrylamide, a cross-linked polyacrylate, polyacrylamide, a copolymer of acrylic acid and acrylamide, or a polyether.

4. The water-absorbing polymer material is crosslinked to form a three-dimensional network structure polymer, and the crosslinking method is physical crosslinking, chemical crosslinking, or physical-chemical mixed crosslinking; The liquid sample adsorption and solidification layer for microbiological testing described in claim 3, wherein the crosslinking that forms the polymer with a three-dimensional network structure is characterized by forming a gel by blending two components or mixing multiple components of water-absorbent polymers, and the mixed colloid is xanthan gum, guar gum, hydroxypropyl guar gum, konjac gum, gellan gum, locust bean gum, sodium carboxymethylcellulose, sodium carboxymethyl starch, or sodium polyacrylate.

5. A polymer having a three-dimensional network structure formed in the liquid sample adsorption / solidification layer for a microorganism test according to claim 4, the water-absorbing polymeric material forms a gel upon addition of a cross-linking agent; The crosslinking agent is a transition metal crosslinker which is aluminum, chromium, titanium, zirconium or a Group IV metal compound; an organotransition metal crosslinker which is an organozirconium crosslinker or an organotitanium crosslinker; Boron crosslinker organic boron crosslinker which is borax or boric acid; A polymer having a three-dimensional network structure, characterized in that it contains any one of the chemical crosslinkers, which are glutaraldehyde crosslinker, formaldehyde, N,N'-methylenebisacrylamide (MBA) crosslinker, genipin, or ethylene glycol diglycidyl ether.

6. A consumable used in the method of claim 1, characterized in that the consumable is one or more of a nutrient necessary for the growth of microorganisms, a microbial growth regulator, a hemolytic agent, a dissolving agent, an antibiotic neutralizer, or a bacterial selective growth agent.

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