CN121006391A - Microorganism detection method and device for large number of liquid samples - Google Patents

Abstract

A method for separating and culturing microbes from a large number of liquid samples includes such steps as using conventional agar culture medium, pouring or dripping quantitative liquid sample to be detected onto the surface of agar culture medium, spraying the aseptic polymer powder onto agar culture medium by dry powder sprayer, cross-linking to form three-dimensional adsorption layer, quickly fixing the microbes on the surface of agar culture medium, incubating the microbes on solidified layer to form bacterial colonies, and counting bacterial colonies. According to the method, a large amount of liquid samples are directly subjected to solid treatment on the basis of a traditional solid culture scheme, so that the integration of the samples and the agar culture medium is realized, and a large amount of liquid samples can be cultured on the agar culture medium.

Description

Microorganism detection method and device for large number of liquid samples

Technical Field

The invention relates to a microorganism detection method for inoculating a large amount of liquid samples, and belongs to the technical field of microorganism culture.

Background

Conventional microorganism culture detection techniques can be classified into agar solid culture methods and broth liquid culture methods. The two methods have advantages and disadvantages, and can not be replaced and only be supplemented. The agar plate streaking method is the most common method for detecting microorganisms, and the microorganisms are determined by smearing or "streaking" a sample on the surface of a gel plate medium, incubating the sample, and then observing whether colonies are present at the streaked portion of the plate. Its advantages are direct taking out colony for microbial identification and drug sensitivity test, but small inoculated sample, and not being able to detect a large number of liquid samples (such as blood culture). The broth culture method has large inoculated sample amount and high positive rate, but can not be used for quantifying bacteria and separating and purifying bacteria.

Microbiological tests on large sample volumes of liquid samples are often required in microbiological work, such as microbiological tests on whole blood (or parts) from clinical microbiological laboratories, on other body fluid samples, microbiological tests on samples on food, pharmaceutical, water and object surfaces in food, pharmaceutical and environmental microbiological laboratories. Therefore, in practical work, a microbiological detection scheme which combines the advantages of the two methods is highly desirable.

In U.S. patent 4,182,656, ahnell et al, a large number of liquid samples can be inoculated into a vessel containing a medium comprising a carbon 13-labeled fermentable substrate. When bacteria are cultured, the ratio of carbon 13 to carbon 12 in the container is measured, and the purpose of measuring the growth of bacteria is achieved by comparing the ratio with the value at the initial stage of the culture. In U.S. patent nos. 4,152,213 and 4,073,691, bacteria are detected by monitoring the gas pressure in the container. In U.S. patent No. 5,094,955, calandra et al, a non-invasive method of detecting the presence of microorganisms in clinical samples (e.g., blood or other body fluids) and non-clinical samples is disclosed wherein a liquid sample is inoculated into a transparent sealed container containing a culture medium, and when the microorganisms are present, the microorganisms grow to produce carbon dioxide, which alters the sensors immobilized within the container, and by analysis of the receptors outside the bottle, non-radiative and non-invasive means of detecting the microorganisms in the presence of interfering substances (e.g., high concentrations of red blood cells) can be achieved.

At present, most commercial liquid sample microorganism culture instruments adopt the detection principle of Calandra et al, but the method also belongs to a liquid broth culture method. Such methods all require special equipment and consumables such as sensors and receptors for observing the growth and metabolism of bacteria, and in addition, the culture vessel must be shaken during the culture process, so that the cost for producing the equipment is high, and the possibility of mechanical failure is increased. Furthermore, such an instrument can only detect the presence of microorganisms, and although the time to positive is related to the number of microorganisms in the sample, it is not possible to count the bacteria. The biggest problem is that after detection of microorganisms, bacteria identification and bacterial drug susceptibility testing are typically required, and the agar solid culture method must be used to isolate pure colonies from the liquid medium to ensure reliability of the susceptibility testing or bacteria identification. This requires additional time (critical patients, life-threatening prolonged detection time) and fails to meet clinical timeliness requirements.

The pour plate method (Pour plate method) is a solid culture method that is currently commonly used to achieve large sample volumes of liquid samples. The conventional liquid sample inoculation amount is about 1-2ml, the requirement of large sample amount such as blood culture and the like cannot be met, meanwhile, the culture medium is required to be prepared and autoclaved in advance during each detection experiment, and the constant temperature of 50 ℃ is kept for standby, so that the preparation is complex and uncontrollable.

The solid plate surface coating method can only inoculate very small liquid samples, about 0.1ml, even on semi-solid media (e.g., agar), and can only absorb less than 5% of the initial gel volume due to the inability of the culture to expand significantly.

Methods for detecting bacteria by membrane filtration can capture microorganisms from a larger volume of liquid, but have a number of drawbacks including increased manual handling time, high material costs, risk of contamination, and difficulty in handling samples containing particulates.

Hyman et al in U.S. Pat. No. 7,183,073 have improved the water absorption properties of solid media by optimizing and modifying the solid culture support to achieve the objective of inoculating large amounts of liquid samples. The culture device contains a polymer immobilization layer having interstitial spaces between the polymer gel matrices. The fluid sample is applied to the immobilized layer, wherein the fluid is absorbed by the layer and the microorganisms remain on the surface of the layer. Although it is described that the liquid absorption amount can reach about 2.5-5ml, the whole absorption process takes more than 10-20 hours. Because the liquid suction is slow, the solid plate can not be turned over, and environmental microorganism pollution is easy to occur in the culture process. Meanwhile, the polymer fixing layer is obviously expanded and deformed after absorbing a large amount of liquid, and the solid gel structure is destroyed, so that the detection of microorganisms is affected. In addition, the proportion of nutrient substances required for the growth of microorganisms is unstable along with the change of the cultured liquid, and the detection effect of the microorganisms is affected. It also cannot use conventional solid agar medium systems. The manufacturing process is complex, and high pressure, vacuum pumping, photo-ultraviolet irradiation, polymerization initiation and the like are needed.

Disclosure of Invention

Problems to be solved by the invention

In the present invention, a large-volume liquid sample containing microorganisms is poured or dropped onto the surface of an agar medium by a sterile method. The liquid in the sample is quickly absorbed by sprayed polymer dry powder, and the high polymer molecules are crosslinked through side chains to form stable gel, so that a liquid sample adsorption solidification layer in a compact hydrogel shape is formed on an agar plate solid culture medium in a short time, microorganisms in the sample are kept in the solidification layer on the surface of the agar plate, the agar plate is turned over, and after bacterial incubation is carried out according to a traditional scheme, single microorganism colonies are easily separated and cultured in the solid culture medium, and the method is convenient for further microorganism detection.

The device consists of a dry powder spraying instrument, liquid solidified dry powder consumable materials (various high polymer materials) packaged by a closed container, a matched sample container and the like.

The dry powder spraying instrument consists of a computer control system, a culture dish mechanical conveying system, a dry powder loading and quantifying device and a dry powder mixing and spraying system. The method comprises the steps of S1 inoculating a large amount of liquid samples (quantitatively pouring or dripping in an aseptic method) onto an agar culture medium, S2 placing an agar culture dish on an instrument, automatically conveying the agar culture dish into the instrument to open the culture dish, S3 selecting a specified amount of aseptic polymer dry powder according to the liquid amount to spray the aseptic polymer dry powder onto the agar culture medium, S4 spraying the aseptic polymer dry powder onto the agar culture medium by using a dry powder mixing spraying device, cross-linking the polymer to form a three-dimensional reticular adsorption layer, S5 fixing microorganisms on the surface of the samples, S6 fixing the microorganisms in the samples on the surface of the agar culture medium after spraying, S6 automatically conveying the agar culture dish outwards after spraying is finished, covering the culture dish on the inner side of the instrument in a moving process, and automatically withdrawing the culture dish from the instrument. And S7, culturing microorganisms in the solidified layer through incubation to form colonies for detection.

The invention realizes sample operation from 0.5ml to 10ml of different liquid amounts. The whole procedure ensures aseptic manipulation. The reagent container is aseptically packaged in a closed way, and the instrument dry powder pipeline system is closed.

The hydrogel-forming polymers used in the present invention may be natural or artificial, but all have common physical properties, and are characterized by 1) rapid absorption of fluid from aqueous solutions or suspensions, swelling of the polymer without dissolution, 2) formation of gels or highly viscous solutions, prevention of the overall fluidity of the fluid, and satisfaction of the conditions for the detection of microorganisms, 3) formation of three-dimensional network structures of chain polymers by compounding of different polymers, or addition of crosslinking agents, etc., and formation of colloids at room temperature, 4) no influence on the growth of microorganisms.

The high molecular polymer used in the invention has enough capability of absorbing water and can absorb a large amount of liquid from a sample. The three-dimensional polymer colloid is formed by moderate crosslinking through mixing or adding a crosslinking agent, so that not only the water absorption is ensured, but also the colloid performance and the liquid absorption speed of the sample, which cannot flow and drip, are ensured. Liquid absorption is usually completed within a period of from a few minutes to 1 hour, so that the problem that the formed colonies have no flow diffusion when the culture dish is turned over for a short time to perform conventional incubation is ensured.

The sample container is a sterile anti-seepage closed container. In order to cooperate with the effect of detecting microorganisms, one or more of nutrient substances, microorganism growth regulator, hemolysis agent, lysing agent, antibiotic neutralizer and bacteria selective growth agent required by the growth of microorganisms can be added into the matched sample container.

The bulk fluid sample of the present invention may be whole blood (or some fraction), other bodily fluids, manufacturing fluids, food samples, and like aqueous liquids.

Drawings

FIG. 1 is a schematic diagram of the principle of microbiological detection of a large number of liquid samples

FIG. 2 is a schematic diagram of a dry powder spray apparatus

FIG. 3 is a graph showing the results of bacterial culture after a large amount of liquid sample is adsorbed by the double gel compounded dry powder to form a solidified layer

FIG. 4 is a graph showing the results of the formation of a solidified layer by adsorbing a large amount of blood sample according to scheme 2 of the multi-gum compound dry powder

FIG. 5 is a graph showing the growth of Staphylococcus aureus in blood samples for 48 hours before and after treatment with adsorbed antibiotics

Detailed Description

FIG. 1 is a schematic diagram of the principle of the method 80, which is a conventionally used agar medium, the method is not limited to the variety of the medium, any agar medium used in the field of microbiology can be used, the air source 10 sprays dry polymer dry powder into a culture dish inoculated with a large amount of liquid sample through air flow control, the polymer can quickly absorb the liquid in the sample, and forms a three-dimensional structure at room temperature to form a hydrocolloid, and a firm solidified layer is formed on the upper surface of the agar medium, and the performance of the solidified layer helps to fix/absorb the sample, so that microorganisms locally grow like the conventional agar culture to form colonies, which increases the capability of detecting microbial colonies. 10, 20, 30, 40, 50, 60, 70, 80 are shown as gas source, mixing chamber, high molecular polymer, gas pressure, atomized dry powder, petri dish, liquid sample, and agar medium, respectively.

Composition of dry powder spray apparatus fig. 2 schematic diagram of dry powder spray apparatus

The dry powder spray apparatus is comprised of a computer control system (not shown), a petri dish mechanical delivery system 100, a dry powder loading and dosing device 200, and a dry powder mixing and spraying system 300.

The dry powder loading container is required to be closed, moisture-proof and sterile. The dry powder loading container of the dry powder loading and dosing device 200 may be connected to the container containing the dry powder reagent via an interface, but the connection is required to be closed. The reagent container filled with the dry powder reagent is designed into airtight sterile package, one end of the reagent container is provided with an aluminum plastic packaging film, after the instrument is connected, the film puncturing device punctures the aluminum plastic packaging film, the reagent container is communicated with the dry powder loading container, and the reagent container can be replaced by a clamp directly after the dry powder reagent is consumed, so that the reagent container can be used only once.

The quantitative device (quantitative adjustment device) of the dry powder loading and quantitative device 200 is provided with a dry powder quantitative hole, and the dry powder reagent in the reagent compartment is quantitatively transported from the dry powder loading container to the dry powder blowing compartment of the dry powder mixing and spraying system 300 by using the size of the quantitative hole. The quantitative device is designed into a volumetric hole or a plurality of holes with the same volumetric hole, and different amounts of the added and dried powder are achieved by controlling the loading times of the quantitative device. That is, a predetermined amount of dry powder may be added according to the single-hole capacity corresponding to any of the plurality of dry powder amounts. In addition, an amount of dry powder of any multiple of the single well volume can be added. The weight of the dry powder may be used to cause the dry powder to naturally drop from the dry powder dosing container into the dry powder dosing aperture. Thereafter, the plate containing the agar medium is moved to the dry powder mixing spraying system 300 by a dish mechanical conveyance system, and the dry powder is sprayed onto the surface of the plate using a gas mixing flow. The dry powder is sprayed by the air flow of the dry powder mixing spray system 300 uniformly spread on the surface of the flat plate.

The whole process is carried out in a closed space, the culture dish is automatically conveyed into the instrument through the culture dish mechanical conveying system 100, meanwhile, the culture dish is opened in the instrument, after the dry powder spraying is finished, the culture dish is covered on the inner side of the instrument, and the culture dish is automatically conveyed out of the instrument. In this way, contamination can be prevented.

< Selection of superabsorbent Polymer >

The high water absorption material is a hydrophilic high polymer material. Common superabsorbent polymers can be classified into natural and modified polymeric superabsorbent materials and synthetic superabsorbent materials.

The natural and modified polymer super absorbent materials are obtained by taking plants, animals or microorganisms as raw materials and extracting by a physical or physicochemical method. The following five categories can be distinguished according to the source of the raw materials:

Vegetable polysaccharide gum is a biopolymer with thickening effect from root, stem/trunk, leaf, seed and fruit of plant, and common vegetable gum is selected from blueberry gum, konjac gum, locust bean gum, carrageenan, guar gum, tamarind gum, gum arabic, etc. Animal protein/polysaccharide gum is prepared from animal skin, bone, tendon, milk, etc. and contains protein biological polymer such as chitosan, gelatin, casein, whey protein concentrate, fish gelatin, etc. The microbial polysaccharide gum is a biopolymer with thickening effect obtained from microbial metabolites, such as xanthan gum, gellan gum, zymosan, etc. Algal polysaccharide gum is a thickening biopolymer extracted from seaweed, such as agar, alginic acid (salt), propylene glycol alginate, red algae gum, fucoidan.

The chemical modified glue is a new glue obtained by processing the natural glue based on the above mentioned structure modification, chemical synthesis and the like, such as methyl cellulose, modified starch, soluble starch, hydroxymethyl cellulose, modified chitosan and the like.

The natural super absorbent materials are commonly natural starch, vegetable gum, animal gum (casein), pectin, chitin, water-soluble derivatives of alginic acid, etc. Such as guar gum, locust bean gum, tara gum, locust gum, tamarind gum, karaya gum, acacia gum, pedicel gum, tragacanth gum, carrageenan, succinan, xanthan gum, gellan gum, and gum reducing. Other natural product lines, alginic acid, chitosan, agar, agarose, soy protein, gluten, sericin.

The semisynthetic modified super absorbent material is prepared by chemical modification of the natural substances. Examples of the starch system include sodium carboxymethyl starch, hydrolysis products of starch graft acrylonitrile, starch graft acrylate polymers, starch graft acrylamide polymers, starch graft styrene sulfonic acid polymers, and starch xanthate graft acrylate. Examples of the cellulose include carboxymethyl cellulose, cellulose grafted acrylate polymer, cellulose sulfoacid salt grafted acrylate, cellulose grafted acrylamide polymer, and cellulose grafted acrylonitrile hydrolysate. Examples of guar gum include hydroxyethyl guar gum, hydroxypropyl guar gum, carboxymethyl hydroxyethyl guar gum, and carboxymethyl hydroxypropyl guar gum.

The synthetic water-absorbing material comprises polyvinyl acid salt, such as cross-linked polyacrylate, polyacrylamide, hydrolysis product of acrylic acid-vinyl acetate copolymer, acrylic acid-acrylamide copolymer, sodium acrylate-vinyl alcohol copolymer (copolymer of methyl acrylate and vinyl acetate), polyacrylonitrile hydrolysis product, polymethyl methacrylate, cross-linked polyacrylate, polyacrylamide, hydrolysis product of acrylic acid-vinyl acetate copolymer, copolymer of acrylic acid and acrylamide, sodium acrylate-vinyl alcohol copolymer (copolymer of methyl acrylate-vinyl acetate), polyacrylonitrile hydrolysis product, and polymethyl methacrylate. The polyvinyl alcohol system is an elastomer obtained by freezing and thawing a polyvinyl alcohol-anhydride cross-linked copolymer, a polyvinyl alcohol-acrylate graft copolymer, a vinyl acetate-acrylate cohydrolysis product, a vinyl acetate-maleic anhydride copolymer, cross-linked polyethylene glycol and polyvinyl alcohol. Polyoxyethylene-based polyethers.

Although super absorbent polymers all have a thickening effect, only a few super absorbent polymers have gel-forming properties in fact. The aqueous solution of the gel-forming polysaccharide can form gel, such as agar, carrageenan, gelatin and the like, and most of the super absorbent polymers can not naturally form gel, such as tamarind gum, locust bean gum, konjak gum, xanthan gum, guar gum and the like.

The gel action is that colloid particles or polymers in sol or solution are combined with each other under certain conditions to form a space network structure (three-dimensional network structure), so that the flow of the system is prevented, and a special dispersion system formed by liquid serving as a dispersion medium is filled in a structural gap.

Hydrogels are three-dimensional polymer networks that contain large amounts of water. The polymers in the network can be associated with large amounts of water due to the presence of hydrophilic functional groups such as-OH, -CONH2, -CONH and-S03H. Therefore, the hydrogel can be kept in a swollen state in water without dissolving. Hydrogels are of a wide variety and can be classified according to different criteria. Depending on the type of crosslinking of the hydrogel, three types of physical crosslinking, chemical crosslinking, and a mixture of both can be classified. Physical hydrogels are generally formed by physical interactions such as ionic, hydrogen bonding, entanglement of chains, and the like. Chemical gels are three-dimensional polymeric network polymers formed by covalent crosslinking that are more stable than physical gels in performance, also known as permanent gels. The hydrogel produced by initiating polymerization of the monomer and the cross-linking agent is generally chemical gel.

Conventional hydrogels are prepared by methods of ① free radical initiated polymerization by ionizing radiation, ② formation of intertwined polymers by chemical reaction, ③ physical interactions such as entanglement, static electricity and crystallite formation.

The physical crosslinking method is to form a three-dimensional network structure of the hydrogel polymer by temporarily and physically crosslinking the hydrogel through the physical acting forces among macromolecules, such as electrostatic action, hydrophobic action, hydrogen bonding action and inter-chain entanglement. Through these forces the polymer is able to interact with multivalent ions, polyelectrolytes, hydrophobic groups, etc. to form a physical gel. Although the preparation method is relatively simple and easy to operate, the gel prepared by the method has poor mechanical properties and is easy to damage, and can be gradually decomposed along with the change of external conditions, and the gelation process is generally reversible. Typically, the physical gel is a polyvinyl alcohol hydrogel, and the aqueous solution of polyvinyl alcohol can form a hydrogel through repeated freezing and thawing processes, and the hydrogel can be de-crosslinked and changed into an aqueous solution under the heating condition. Agarose dissolves in water at high temperature to form a solution, which crosslinks to form a network when cooled, forming a gel. And (3) radiation crosslinking. Some polymers are exposed to high energy radiation (e.g., gamma rays or electron beams) and the polymer chains will spontaneously crosslink by radiation-induced free radicals. Sodium alginate can be crosslinked into hydrogel through Ca2+, and gelatin and agarose can also form hydrogel under the action of hydrogen bonds.

Chemical gels are hydrogels formed by intermolecular cross-linking through covalent bonds, and general synthetic gels are of this type. Such covalent crosslinks are typically formed by copolymerization of monomers with crosslinking agents or by interaction of functional groups in the molecular chain. The covalent crosslinks are very strong and the gelation process is generally irreversible.

The cross-linking mode searched by the invention meets the following requirements that under the condition of room temperature, no additional steps such as heating, high pressure or irradiation are needed, moderate cross-linking is generated in a short time, and the gel is formed while the relatively large water absorption performance is ensured.

The high molecular polymer with high water absorption performance is a long-chain polymer, the network crosslinking is less, and when the substance is crosslinked into a network structure, the water absorption capacity is reduced and water is separated out. Therefore, it is critical to control the degree of crosslinking, and gels formed from polymers must maintain cohesion or high viscosity under the conditions of use to maintain water absorption and crosslink to form colloids. The gel must remain intact during the volume and temperature changes required for its use. It is also desirable that the polymer is not readily degraded by the microorganism being cultured. Furthermore, the inter-chain cross-linking or entanglement of the gelling polymer must be high enough to maintain gelling or high viscosity, but low enough to allow for high swelling. More specifically, whatever material is used (natural, synthetic or semi-synthetic polymers, or other materials), it is preferable to provide a network of mutually bonded polymer chains that are flexible enough to absorb liquids without disrupting the network.

Common crosslinking agents are transition metal crosslinking agents such as aluminum, chromium, titanium, zirconium or group IV metal compounds, organic transition metal crosslinking agents such as organozirconium crosslinking agents, organotitanium crosslinking agents, boron crosslinking agents such as borax, boric acid and organoboron crosslinking agents, chemical crosslinking agents such as glutaraldehyde crosslinking agents, formaldehyde, N' -Methylenebisacrylamide (MBA) crosslinking agents, genipin, ethylene glycol diglycidyl ether and the like.

< Double gel Compound gel >

The double-gel compound gel is a gel formed by blending (compounding) two water-absorbing high polymer polysaccharide

Some colloids (water-absorbing polymers) do not form gels alone at room temperature, and such gels are known as non-curdlan, but non-curdlan forms a gel when compounded with some other polysaccharide gums.

Xanthan also has a higher viscosity at lower concentrations, is a pseudoplastic fluid, but does not form an elastic gel. Xanthan gum has a significant synergistic effect with many non-curdlan, and the viscosity of the mixed gel is several times higher than that of a single gel at the same concentration or is in a jelly shape, and the phenomenon is called synergistic thickening property and synergistic gel property. This is mainly due to the fact that the double helix structure of the xanthan molecule is susceptible to chimerism with polysaccharide molecules containing beta-1, 4 linkages. With this interaction, the gel strength is maximized. The two polysaccharide high molecular polymers are blended in a proper proportion so as to achieve the maximum possible synergistic effect between the two polysaccharide molecules, and the maximum gel capability and the maximum gel strength are shown.

Also konjak glucomannan (konjak gum) has a high apparent viscosity and does not exhibit gel properties under non-alkaline conditions. However, if xanthan gum is added into the konjac glucomannan solution, the apparent viscosity of the konjac glucomannan and xanthan gum compound gum gradually increases along with the increase of the addition amount of the xanthan gum, and the maximum value is reached when the mass ratio of the konjac glucomannan to the xanthan gum is equal, and then the apparent viscosity gradually decreases. This means that konjak glucomannan and xanthan gum can not only thicken but also have gelling property when they interact with each other in a certain proportion. The konjac glucomannan and the xanthan gum have strong synergistic effect that a double-helix structure of the xanthan gum is not formed at the joint of a branched chain and a secondary bond, a three-dimensional network structure is formed on the part of the konjac glucomannan molecules with smooth molecules, when the concentration of the compound gum reaches a certain value, elastic gel is formed, and the gel strength is increased along with the increase of the concentration.

When the mixing ratio of the xanthan gum to the konjak glucomannan is 70:30 and the total concentration of the polysaccharide is 1%, the maximum value of the synergistic interaction can be reached, and a solid gel is formed.

< Multi-gel Mixed gel >

Multi-gel compounding refers to the mixing of two or more gels having different properties due to differences in chemical composition and structure. When these different substances are present at the same time, their properties tend to change to different extents due to interactions and influences between them. Compared with monomer glue, the mixed glue has obvious glue forming advantage.

Through mixing, the complementary effect of various single glues can be exerted, thereby expanding the application range of the glue or improving the application performance of the glue. The synergistic effect can enhance the rheological property of the colloid, control the speed of forming the colloid and improve the uniform and transparent appearance of the colloid.

The locust bean gum, the xanthan gum, the guar gum and the gellan gum have obvious gel synergistic effect.

Locust bean gum itself has no gel character but has a good gel synergistic effect with other hydrocolloids.

Guar gum is a swelling polymer with extremely high water absorption performance, the structure of guar gum is a coiled spherical structure, and contains a large amount of hydrophilic hydroxyl groups, but most of the hydroxyl groups are in the interior of molecules structurally, and intermolecular forces enable the hydroxyl groups to form intramolecular self-crosslinking. Guar gum and modified guar gum are mixed with certain linear polysaccharides such as xanthan gum, agar gum, K-carrageenan, locust bean gum, sodium alginate, konjac gum, gellan gum and starch to form a gel.

< Stability of Mixed glue >

The stability of hydrogels formed by different types of gums varies, acidic and basic conditions have a great influence on some gels, as the pH changes during bacterial growth, the hydrogels formed melt and various enzymes produced by the bacteria can also dissolve the hydrogels that have formed.

Xanthan gum, konjak gum, gellan gum, guar gum, hydroxypropyl guar gum, cationic guar gum, xanthan gum and konjak gum compound gum and Huang Mohuai melon compound gum are good in acid-base stability and enzyme stability. Is very stable to acid and alkali, and can be used under both acidic and alkaline conditions. The solution has stable viscosity within the pH range of 2-12. Typical microbial or industrial enzymes, such as proteases, cellulases, pectinases, amylases have no effect on these gums. The Huang Mohuai melon is compound mixed glue of 4 kinds of glue. The compound mixed gum is fully called xanthan gum, konjak gum, locust bean gum and guar gum.

< Use of crosslinking agent >

In order to ensure the colloid performance of the liquid solidified layer and enable the water-absorbing high molecular polymer to form a stable three-dimensional network structure, the invention uses a chemical cross-linking agent to detect a large number of liquid sample bacteria.

< Addition of nutrients, regulators, hemolytic agents, lysing agents, antibiotic neutralizing agents, bacteria-selective growth Agents)

In order to improve the effect of microorganism detection, one or more of nutrient substances, microorganism growth regulator, hemolysis agent, lysing agent, antibiotic neutralizer and bacteria selective growth agent required by microorganism growth can be added into the detection system. According to the design, the substances can be added into corresponding containers in advance according to specific test purposes, such as adding an antibiotic neutralizer and an anticoagulant into a blood culture tube to remove factors interfering bacteria growth in a sample, such as cell immunity factors like WBC, antibody complement humoral immunity factors, medicines like antibiotics and the like. The container can be designed as a special container for neutralizing disinfectant for environmental monitoring, a special container for medicine detection, a special container for food detection, and the like.

Wherein the hemolytic agent or lysing agent dissolves or lyses blood cells or other human tissue substances in the specimen, facilitating the absorption of moisture. The hemolytic agent is saponin, digitonin, tween, monolaurate or other surfactants, and the lysing agent is protease.

The microorganism growth regulator may be various growth factors of bacteria, such as tryptone, soytone, peptone, malt extract, glucose, hemin, cystine, disulfide, menaquinone, coenzyme I.

The antibiotic neutralizer can remove the influence of antibiotics in the specimen on bacterial culture, and improve and/or quicken the culture of microorganisms in the test sample. Such as various resins, gums, carbon-based materials (e.g., activated carbon), antibiotic-degrading enzymes (e.g., beta-lactamase).

The bacterial growth promoter is selected from lactose, bile salt, K2HPO4, mgSO4, magnesium sulfate, disulfide, SDS, and fungus and other acid-resistant microorganism, and glucose, yeast extract, NHSiCl4, citric acid or tartaric acid can be added.

< Sterilizing method of consumable Material >

To ensure sterility of disposable consumables such as water-absorbing dry powder reagents or liquid crosslinkers provided to the user, radiation sterilization can be performed on consumables that are not suitable for autoclaving. The irradiation sterilization has no influence on the appearance and performance of various rubber powders, the appearance and performance of the liquid cross-linking agent have no change, and the water sample solution is colorless and transparent. The conventional irradiation dose of 8K cannot achieve the aseptic effect. The dose irradiation dose of 25K is required to be twice or more times every day to ensure the sterilization effect. The consumable may further include one or more of nutrients, microbial growth regulators, hemolysis agents, lysing agents, antibiotic neutralizers, or bacteria selective growth agents necessary for microbial growth, and may be contained in a sample container.

Example 1]

Xanthan gum and konjak glucomannan dry powder

The dry powder is prepared according to the proportion of xanthan gum dry powder to konjak glucomannan dry powder=70:30, fully mixed, placed into a closed container, autoclaved at 121 ℃ and dried at 80 ℃ overnight. And (5) standby.

< Method >

The liquid sample is dripped on an agar culture medium plate, the xanthan gum konjak glucomannan dry powder is uniformly sprayed into the culture plate inoculated with a large amount of liquid sample through high-pressure gas according to the proportion of 4% w/v under the aseptic condition, the high-molecular polymers can rapidly adsorb the liquid in the sample, a three-dimensional structure is formed under the room temperature condition, the liquid sample becomes hydrogel, a firm solidified layer is formed on the upper surface of the agar culture medium, and the performance of the solidified layer is favorable for fixing/absorbing the sample. The saline water can be quickly sucked dry, the plates are turned upside down after 1-10 minutes, and the plates are placed into a 37 ℃ incubator, so that microorganisms can locally grow to form colonies like conventional agar culture, and the conditions of the culture medium and the bacteria growth are continuously observed for 1-14 days.

Observing according to the proportion of 4%

The dry powder was sprayed uniformly with 0.04g per 1ml of brine.

As a result, after overnight incubation, all liquid was absorbed by the gel, bacterial colonies that separated from each other were easily observed on the surface of the gel, and could be directly harvested.

The results are shown in FIG. 3. FIG. 3 shows the result of bacterial culture after a large amount of liquid sample is adsorbed by the double-bonded composite dry powder to form a solidified layer,

Dry powder of double-gum compound fixative (dual gum compound fixer dry powder) is composed of xanthan gum dry powder, konjak glucomannan dry powder=70:30

Nutrient agar culture dish

Liquid sample, non-sterilized saline solution 3ml

Example 2]

Multi-glue dry powder scheme 1 (Huang Mohuai melon 6211 mixed dry powder)

On the basis of the optimized preparation of the two-component dry powder curing agent, the formula of the optimal multi-component dry powder curing agent is searched. The water absorption capacity is improved, the physiological saline can be absorbed by the multi-gel mixed dry powder to more than 10ml, and the excellent colloid performance is ensured. The initial water absorption is quicker, 1-3ml of physiological saline can be absorbed quickly in 1-5 minutes, but when the volume is more than 4ml, the liquid is absorbed in 10-30 minutes, and the culture dish is reversed and placed into an incubator for culture. Solves the stability problem of the hydrogel, the gel formation is stronger in the next day, the performance of the hydrogel is stable and unchanged for a long time (1-14 days), and when bacteria grow, even the pH changes and various enzyme colloids are generated, the gel is stable.

Through mixing and preliminary screening of xanthan gum, guar gum, hydroxypropyl guar gum, konjak gum, gellan gum, locust bean gum, sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium polyacrylate and the like, the four kinds of gums are screened out for mixing and testing, wherein the xanthan gum has the advantages of no colloid formed by single component, uniform and fine particles, strong water absorption, viscosity and low cost. The konjak gum is characterized in that a single component can form colloid, the particles are thicker, the water absorption is stronger, the water absorption is slower, and the dry powder distribution is uneven. Locust bean gum, a single component of which can not form a colloid. The solution is very thin, has good fluidity, is favorable for uniform distribution of dry powder, and brings excellent synergistic effect of lateral branch crosslinking. Hydroxypropyl guar gum is a single component which can not form colloid, has excellent water absorption and can absorb water in a short time. After optimizing the multiple groups of compound combinations, the formula proportion is determined to be 6:2:1:1.

< Formulation of multicomponent Dry powder curing agent >

Xanthan gum, konjac gum, locust bean gum, hydroxypropyl guar = 6:2:1:1. Thoroughly mixed, placed in a closed container, autoclaved at 121 ℃ and dried overnight at 80 ℃. And (5) standby.

< Method >

Liquid samples of different volumes were poured or dripped onto agar medium plates and the dry powder was sprayed in a ratio of 3% w/v under sterile conditions. The water absorption in the culture dish was observed, the liquid was sucked dry by naked eyes, the culture dish was put into a 37 ℃ incubator, and the culture medium and the bacterial growth were continuously observed for 1-14 days.

The firmness of the combination of the solidified layer and the agar medium is observed, and the solidified layer absorbs the sample, and the microorganisms grow and form colonies.

Example 3]

Multi-glue mixing dry powder scheme 2 (Jieqiang 721 mixed dry powder)

Unlike saline, blood contains a large amount of proteins, inorganic salts and various substances, the existence of which affects the formation of the three-dimensional structure of the hydrogel and also affects the stability of colloid performance, and we establish a multi-colloid compounding dry powder scheme 2 for carrying out a blood adsorption curing scheme.

< Formulation of multicomponent Dry powder curing agent >

Gellan gum, hydroxypropyl guar, cationic guar = 7:2:1. Thoroughly mixed, placed in a closed container, autoclaved at 121 ℃ and dried overnight at 80 ℃. And (5) standby.

< Method >

The same procedure as in example 2

The firmness of the combination of the solidified layer and the agar medium is observed, and the solidified layer absorbs the sample, and the microorganisms grow and form colonies.

The results are shown in FIG. 4. Fig. 4 is the result of the multi-glue compounding dry powder regimen 2 adsorbing a large number of blood samples to form a solidified layer.

Compounding hardener dry powder gellan gum, hydroxypropyl guar, cationic guar = 7:2:1

Culture medium type, columbia blood agar plate

10Ml of normal anticoagulated blood

Example 4]

Ultra-large volume liquid sample solidified layer

In the invention, in order to absorb a huge amount of liquid in a sample and ensure the colloid performance of a liquid curing layer, a stable three-dimensional network structure is formed in a water-absorbing polymer, and a chemical cross-linking agent is respectively added into single colloid hydroxypropyl guar and multicomponent compound gum to cure the huge amount of liquid sample (20 ml).

Material hydroxypropyl guar and yellow locust melon 6211 (homemade)

Crosslinking agent organozirconium crosslinking agent (DuPont Tyzor 212)

Sample type of physiological saline and anticoagulated blood

< Method >

Mixing hydroxypropyl guar gum and yellow locust melon 6211 with dry powder, and sterilizing under high pressure. When the injection is carried out in high-pressure sterilization, the sealing and moisture prevention are needed. The blood plate agar culture medium is added with 20ml of anticoagulated blood specimen (or normal saline) +organozirconium cross-linking agent, dry powder is sprayed from above, the mixture is kept stand for 1 hour at 37 ℃, the culture medium is turned over, and the mixture is placed in a 37 ℃ incubator for culture, and is observed for 3-5 days.

The results are shown in Table 1

Table 1 generation of gel in solidified layer of 20ml physiological saline sample

Table 2 gel formation of 20ml anticoagulated blood sample solidified layer

Example 5]

Effect of organozirconium Cross-linking Agents on bacterial growth

The effect of the organozirconium cross-linking agent on bacterial growth was observed. Determining whether the organozirconium cross-linking agent can be used for bacterial detection, and selecting the safe use amount of the cross-linking agent.

Material 1. Organozirconium crosslinker (DuPont Tyzor 212). 2. French biology Mei Liai company blood culture bottle (child bottle 30 ml). 3. The clinical isolated strain is fresh prepared bacterial liquid for clinical drug sensitivity test.

< Method >

Firstly, preparing bacterial liquid by using physiological saline, detecting by using a turbidimeter, wherein the bacterial liquid concentration is 0.5 McUth (10 ⁸ cfu/ml), diluting the bacterial liquid, and finally preparing bacterial liquid with the concentration of 3 cfu/ml. Two French living beings Mei Liai blood culture bottles are taken as a measuring tube and a control tube respectively, and the bacterial liquid is respectively injected into the two French living beings Mei Liai blood culture bottles, wherein the inoculation amount is 1 ml/bottle, and 0.9ml (0.3% (v/v) of 10% organic zirconium cross-linking agent is added into the measuring bottle, so that the preparation is completed within 5 minutes. According to the requirements of a user operation manual of a Mei Liai company blood culture instrument, the blood culture bottle is put into the instrument, and the instrument automatically detects.

The results are shown in Table 3

Table 3 influence of organozirconium crosslinker on bacterial growth

The result shows that the system contains 0.3% (v/v) organic zirconium cross-linking agent, which has no influence on bacterial growth, and can be completely used for conventional microorganism detection work.

Example 6]

Effect of detecting bacteria of clinical blood sample of resin blood-sampling Guan Digao

Clinical patients often have a large number of antibiotics used for a long period of time before blood culture is performed, which severely affects the positive rate of bacterial isolation culture in blood. The antibiotic neutralizer can remove the influence of antibiotics in the specimen on bacterial culture, and improve and/or quicken the culture of microorganisms in the test sample.

The measuring tube is characterized in that the 8ml blood collection tube contains 0.6g of macroporous adsorption resin and SPS anticoagulant, and the 8ml blood collection tube contains only SPS anticoagulant.

The preparation of the simulated positive blood sample comprises the steps of adding staphylococcus aureus ATCC25923 into normal saline to prepare the final bacterial liquid concentration of the simulated sample of 1-10cfu/ml.

4Ml of blood of a clinical antibiotic-treated patient was collected, and 2ml of each of the measurement tube and the control tube was separately injected.

Huang Mohuai melon 6211, and autoclaving. In autoclaving, the container is sealed against moisture. The blood plate agar medium is added with 2ml of the anticoagulated blood sample, dry powder is sprayed from the upper part, the mixture is kept stand for 1 hour at 37 ℃, the medium is turned over, and the mixture is placed in a 37 ℃ incubator for culture.

The results are shown in FIG. 5. FIG. 5 shows the growth of Staphylococcus aureus in blood samples 48 hours before and after antibiotic adsorption treatment,

Dry powder of mixed solidifying agent xanthan gum, konjak glucomannan gum, locust bean gum, hydroxypropyl guar = 6:2:1:1

Culture medium type Columbia blood agar culture dish

Liquid sample 2ml anticoagulated blood

The strain is staphylococcus aureus ATCC25923.

The upper panel a shows 2ml of blood from a patient who has been treated with an adsorption resin and the lower panel B shows 2ml of blood from a patient who has not been treated with an adsorption resin and has been treated with an antibiotic. In order to cultivate the staphylococcus aureus ATCC25923 colony for 48 hours, the result of the measuring tube is obviously better than that of the control tube, which shows that the adsorption resin has obvious effect of removing antibiotics and has good application effect.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Compared with the prior art, the invention has the following advantages:

1. According to the detection method for the microorganisms in the large number of liquid samples, disclosed by the invention, the large number of liquid samples are directly subjected to solid treatment on the basis of a traditional solid culture scheme, so that the integration of the samples and an agar culture medium is realized, and the culture of the large number of liquid samples on the solid agar culture medium is completed. According to the scheme, the traditional culture method that a large amount of liquid samples are subjected to broth culture firstly and then agar solid separation culture is further carried out when the large amount of liquid samples are cultured is solved, so that the culture procedure is simplified, and the culture time and the detection time are saved. The scheme realizes the combination of liquid culture and solid culture, also realizes the combination of bacterial culture and bacterial separation, can also quantify microorganisms, and improves the positive rate of microbial culture.

2. A method and apparatus for separating and culturing microorganisms from a plurality of liquid samples is disclosed. According to the method, a traditional agar culture medium is used, a quantitative liquid sample to be detected is poured or dripped on the surface of the agar culture medium in an aseptic method, aseptic polymer dry powder is sprayed on the agar culture medium by using a dry powder spraying instrument designed for the method, and the polymer is crosslinked to form a three-dimensional reticular structure adsorption layer, so that microorganisms are rapidly immobilized in the sample. The microorganisms in the sample are rapidly immobilized on the surface of the agar culture medium, and the microorganisms in the solidified layer form colonies for detection after incubation and culture, can count the colonies, and can also be directly applied to further operations such as bacteria identification, drug sensitivity test or other microbiological analysis and detection.

3. According to the microorganism detection method, the high water-absorbing material dry powder is sprayed into the liquid sample to absorb a large amount of liquid, meanwhile, the high polymer is utilized to form a three-dimensional network structure, and gel is formed under the room temperature condition, so that the gel has excellent transparency and controllable properties, and the growth of microorganisms is not influenced. The invention has high water absorption speed, can be completed in a few minutes to a few hours, and is favorable for bacteria to cultivate independent bacterial colonies. The pure colony can directly carry out relevant operations such as bacteria identification, drug sensitivity and the like.

4. According to the microorganism detection method, the water absorption capacity is large, the maximum of the 90mm diameter flat plate culture medium can absorb 20mL of liquid, and the common agar culture medium can only absorb 0.2mL of liquid within 30 minutes.

5. The detection method of a large number of liquid sample microorganisms has short culture time, generally not more than 3 days, and can be finished in 18 hours. In the current clinical work, the culture period of liquids such as blood, hydrothorax, ascites, joint effusion, pericardial effusion and the like is usually 5-7 days.

6. The method greatly expands the application range of the solid agar plate culture method. When detecting microorganisms in medicine, industry or food, a great number of liquid samples are usually cultured by liquid broth culture, and the invention provides a brand new scheme.

Claims (6)

1. A method for detecting microorganisms includes providing a device for detecting microorganisms and matched consumable materials, spraying the dried powder of high molecular polymers capable of forming hydrogel onto the surface of an agar culture medium, crosslinking the high molecular polymers to form polymers with three-dimensional network structures, preparing a liquid sample adsorption solidified layer, fixing all microorganisms in the sample on the surface of the agar culture medium, incubating and culturing, and detecting microorganisms in the solidified layer.

2. A device for use in the method according to claim 1, said device being a dry powder spray apparatus, characterized by consisting of a computer control system, a Petri dish mechanical transport system, a dry powder loading and dosing device, a dry powder mixing spray system, said dry powder loading and dosing device being designed with a dry powder dosing aperture on its dosing device, dry powder naturally falling into said dry powder dosing aperture from a dry powder dosing container of said dry powder loading and dosing device by dry powder weight, a plate containing said agar medium being moved by Petri dish mechanical transport system to said dry powder mixing spray system, dry powder being sprayed onto said plate surface by gas mixing flow.

3. A liquid sample adsorption cured layer for microorganism detection used in the method according to claim 1, characterized in that the liquid sample adsorption cured layer contains a water-absorbent polymer material, characterized in that a natural-, semisynthetic-modified-, or synthetic-type superabsorbent material,

The natural super absorbent material is natural starch, vegetable gum, animal gum, pectin, chitin, seaweed derivative, guar gum, konjak gum, locust bean gum, tamarind gum, acacia gum, carrageenan, xanthan gum, gellan gum, alginic acid (salt), chitosan, agar, agarose, gelatin, casein or casein,

The semisynthesis modified super absorbent material is carboxymethyl starch sodium, starch grafted acrylic acid salt polymer, starch grafted acrylic acid amide polymer, carboxymethyl cellulose, cellulose grafted acrylic acid salt polymer, cellulose grafted acrylic acid amide polymer, hydroxyethyl guar gum, hydroxypropyl guar gum, carboxymethyl hydroxyethyl guar gum or carboxymethyl hydroxypropyl guar gum,

The synthetic super absorbent material is cross-linked polyacrylate, polyacrylamide, a copolymer of acrylic acid and acrylamide, or polyether.

4. The liquid sample adsorption cured layer for detecting microorganisms according to claim 3, wherein the water-absorbent polymer material is crosslinked to form a polymer having a three-dimensional network structure by physical crosslinking, chemical crosslinking and physical-chemical hybrid crosslinking,

The polymer forming the three-dimensional network structure is crosslinked, and is characterized in that the gel is formed by double-component compounding or multi-component compounding of a water-absorbing high molecular polymer, wherein the compounded gel is xanthan gum, guar gum, hydroxypropyl guar gum, konjak gum, gellan gum, locust bean gum, sodium carboxymethyl cellulose, sodium carboxymethyl starch or sodium polyacrylate.

5. The polymer of three-dimensional network structure according to claim 4 wherein the water-absorbing polymeric material is gelled by addition of a cross-linking agent,

The cross-linking agent is:

transition metal crosslinkers, i.e.aluminum, chromium, titanium, zirconium or group IV metal compounds,

An organic transition metal crosslinking agent, i.e., an organozirconium crosslinking agent, or an organotitanium crosslinking agent,

Boron crosslinking agent an organoboron crosslinking agent, borax, or boric acid,

Chemical cross-linking agents, i.e., glutaraldehyde cross-linking agents, formaldehyde, N' -Methylenebisacrylamide (MBA) cross-linking agents, genipin, or ethylene glycol diglycidyl ether.

6. A consumable for use in the method according to claim 1, characterized in that the consumable is one or several of a nutrient substance, a microorganism growth regulator, a haemolytic agent, a lysing agent, an antibiotic neutralizer, or a bacteria selective growth agent required for the growth of microorganisms.