KR20030096986A - Identification composition for non-fermentative gram negative bacilli and identification kit containing the same - Google Patents

Abstract

PURPOSE: A composition and kit for identification of non-fermentative gram negative bacteria are provided, thereby identifying bacteria in the spleen, and diagnosing diseases caused by bacteria. CONSTITUTION: A method for identifying non-fermentative gram negative bacteria comprises the steps of: (a) preparing suspension of a microorganism to be identified; (b) reacting the suspension with compositions for testing sugar oxidization, beta-galactosidase activity, gelatinase activity, esculin hydrolysis, organic acid alkalization, urease activity, arginine dihydrolase activity, hydrogen sulfate production reaction, fermentation, nitric acid reduction, and indol production; and (c) analyzing the results of reactions. A composition for identification of non-fermentative gram negative bacteria comprises NH4H2PO4, MgSO4 7H2O, KCl, yeast extract, casamino acid, polypeptone, trace undiluted urine solution, CaCl2, undiluted vitamin solution, ferric ammonium salt citrate, BTB and sugars, wherein the trace undiluted urine solution comprises sodium molybdate, boric acid, cobalt chloride, zinc sulfate, manganese chloride, sodium selenite, nickel chloride and CuCl2; and the undiluted vitamin solution comprises vitamin B12, biotin, folic acid, riboflavin, nicotinic acid, lipoic acid, ρ-aminobenzoic acid, thiamine, calcium DL-pantothenate and pyridoxine.

Description

Non-fermented Gram negative bacteria identification composition and identification kit using the same {IDENTIFICATION COMPOSITION FOR NON-FERMENTATIVE GRAM NEGATIVE BACILLI AND IDENTIFICATION KIT CONTAINING THE SAME}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a non-fermentative Gram-negative bacterium identification composition and identification kit using the same. More particularly, the present invention relates to a test composition, identification method, and identification kit that can identify 24 kinds of biochemical characteristics of microorganisms at once.

[Private Technology]

Identification of microorganisms refers to the identification and identification of taxonomic locations using biochemical, morphological, chemical or molecular biological properties of unknown microorganisms.

Classic microbial identification methods include AFB staining, Albert staining, Bacitracin test, Bile-escμlin test, CAMP test, Catalase test, and citrate CITRATE UTILIZATION, DECARBOXYLASE test, DNase test, Gram staining, India ink Preparation, Indole test, 10% KOH (NaOH) preparation, Methyl red test, MSA (Mannitolsalt agar) Tests, 6.5% NaCl test, NIACIN test, nitrite test, Optochin test, Oxidase test and Voggs-Proskauer test are conducted.

Based on this method, a variety of microorganism identification kits have been developed and are commercially available. For example, API 20 E (API 20 Enterobacteriaceae), API 20 NE (API 20 Non-Enterobacteriaceae), API 20 STREP (API 20 Streptococcus), API STAPH (API Staphylococcus), API 20 C AUX API 20 candida, API NH ( API Neisseria and Haemophilus (API), API Listeria (API Listeria), API Coryne (API Coryne), ID 32 GN (Identification system for Gram negative rods), ID 32 C (Identification system for yeasts), and the like. It is designed to identify only bacteria.

A recently developed identification method is a method for identifying ribosomal rRNA by PCR. However, this method has to establish the rRNA information of each bacteria, not only hassle of having to perform several PCR processes, but also has a problem that it is difficult to accurately analyze the mutant species.

On the other hand, microbial identification is a process that must be performed to identify pathogens or to identify microorganisms useful in a new or specific environment. However, many experiments must be carried out to identify microorganisms, and thus, a lot of time and money are required to identify a strain.

Therefore, it is necessary to develop a method for easily and simply identifying and identifying various characteristics of microorganisms.

In order to solve the problems of the prior art, an object of the present invention is to provide a method that can easily and simply identify non-fermented Gram-negative bacteria.

In addition, the present invention is a sugar oxidation reaction, beta-galactosidase activity, gelatinase activity, esculin hydrolysis reaction, organic acid alkalizing reaction, urease activity, arginine dehydrolase activity, hydrogen sulfate production reaction, fermentation, An object of the present invention is to provide a composition for identification, which can identify the nitric acid reduction reaction and the indole production reaction, respectively.

Another object of the present invention is to provide an identification kit that can identify non-fermented Gram-negative bacteria using the composition for identification.

In order to solve the above technical problem, the present invention is a sugar oxidation reaction, beta-galactosidase activity, gelatinase activity, esculin hydrolysis reaction, organic acid alkalizing reaction, urease activity, arginine dehydrolase activity for the microbial suspension It provides a method for identifying non-fermented Gram-negative bacteria by checking the hydrogen sulfate production reaction, fermentation, nitric acid reduction reaction and indole production reaction, respectively.

The present invention also provides a composition for testing sugar oxidation reaction, a composition for testing beta-galactosidase activity, a composition for testing gelatinase activity, a composition for esculin hydrolysis reaction, a composition for testing organic acid alkalizing reaction, and a urease activity test. It provides a composition for identification, comprising a composition for the test, arginine dihydrolase activity test composition, hydrogen sulfate production reaction, fermentation test composition, nitric acid reduction test composition and indole production test composition.

In addition, an object of the present invention is to provide a kit for identification comprising at least one composition selected from the group consisting of the test compositions.

Hereinafter, the present invention will be described in detail.

The present inventors selected only essential methods that can easily identify only non-fermented Gram-negative bacteria among various identification methods, and optimally prepared the test compositions for each identification method in order to increase the accuracy of each identification result.

Identification method using the sugar oxidation reaction of the present invention is to distinguish the oxidizing power of a specific sugar of the microorganism, glucose, lactose, sucrose, mannitol, maltose, mannose, rhamnose, galactose, fructose, xylose and arabinose It is to check the oxidation of the. When oxidation occurs, the pH in the medium is lowered. This phenomenon is confirmed by the color change of the pH indicator. Examples of pH indicators that can be used for the sugar oxidation reaction is bromothymol blue sodium salt (BTB), and BTB is yellow when a sugar oxidation reaction occurs.

In order to confirm the above-mentioned sugar oxidation reaction, a sugar usability test composition is prepared.

Sugar composition for usability testing

(a) NH ₄ H ₂ PO ₄ , MgSO ₄ · 7H ₂ 0, KCl, yeast extract, casamino acid, polypeptone, soypeptone, trace urea stock solution, CaCl ₂ , vitamin stock solution, ferric ammonium citrate, BTB Oxidation reaction medium comprising; And

(b) at least one sugar selected from the group consisting of glucose, lactose, sucrose, mannitol, maltose, mannose rhamnose galactose, fructose, xylose and arabinose and having a pH of 7.0 to 8.0. The most preferred pH is 7.6.

The trace stock solution medium is sodium molybdate (Na ₂ MoO ₄ · 2H ₂ O), boric acid (H ₃ BO ₃ ), cobalt chloride (CoCl ₂ · 6H ₂ O), zinc sulfate (ZnSO ₄ · 7H ₂ O), Manganese chloride (MnCl ₂ · 4H ₂ O), sodium selenite (Na ₂ SeO ₃ ), nickel chloride (NiCl ₂ ) and cupric chloride (CuCl ₂ · 2H ₂ O), wherein the vitamin stock solution is vitamin B12 , Biotin, folic acid, riboflavin, nicotinic acid, lipoic acid, p-aminobenzoic acid, thiamine, calcium DL-pantothenate and pyridoxine.

The composition for testing sugar availability of the present invention more preferably

(a) 1 to 0.01 wt% NH ₄ H ₂ PO ₄ , 1 to 0.0001 wt% MgSO ₄ · 7H ₂ 0, 1 to 0.001 wt% KCl, 1 to 0.0001 wt% yeast extract, 1 to 0.0001 wt% % Cassanoic acid, 1 to 0.0001 wt% poly peptone, 1 to 0.0001 wt% soypeptone, 10 to 0.1 wt% trace urea stock, 1 to 0.00001 wt% CaCl ₂ , 1 to 0.01 wt% vitamin stock 90-97 wt% of an oxidation medium comprising 1-0.0001 wt% ferric ammonium citrate, 1-0.0001 wt% BTB and 90-99.9999 wt% water and

(b) 3 to 10% by weight per sugar selected from the group consisting of glucose, lactose, sucrose, mannitol, maltose, mannose rhamnose galactose, fructose, xylose and arabinose.

The trace urea stock solution also contains 1 to 0.001 wt% sodium molybdate, 1 to 0.001 wt% boric acid, 1 to 0.001 wt% cobalt chloride, 1 to 0.001 wt% zinc sulfate, 1 to 0.0001 wt% manganese chloride Preferably, 1 to 0.0001 wt% sodium selenite, 1 to 0.0001 wt% nickel chloride, 1 to 0.0001 wt% cupric chloride, and 92 to 99.999 wt% distilled water,

The vitamin stock solution is 0.01 to 0.000001 wt% vitamin B12, 0.1 to 0.00001 wt% biotin, 0.1 to 0.00001 wt% polyacid, 0.1 to 0.00001 wt% riboflavin, 0.1 to 0.00001 wt% nicotinic acid, 0.1 to 0.00001 wt% Lipoic acid, 0.1-0.00001% by weight of p-aminobenzoic acid, 0.1-0.00001% by weight of thiamin, 0.1-0.00001% by weight of calcium DL-pantothenate, 1-0.0001% by weight of pyridoxine and 98-99.999% by weight It is preferable to contain distilled water of.

The sugar usability testing composition of the present invention changes the composition color from blue to yellow when a sugar oxidation reaction occurs. Therefore, when a microorganism sample reacts with the composition for testing glucose availability, the composition turns yellow, indicating that any microorganism used is a bacterium that uses glucose as an energy source.

The present invention also provides an identification method and a test composition using beta-galactosidase activity. The identification method is a method for screening microorganisms expressing beta-galactosidase, and is identified by using a color of a compound decomposed by beta-galactosidase. A representative example of the compound is ONPG (ο-nitrophenyl-β-D-galactopyranoside), which ONPG is degraded by beta-galactosidase to give yellow color.

The beta-galactosidase activity test composition of the present invention includes bacto peptone, Na ₂ HPO ₄ , ONPG and distilled water. A preferred content of the composition for assaying beta-galactosidase activity is (a) a peptone medium comprising 0.1 to 10% by weight of bacto peptone and 90 to 99% by weight of distilled water and (b) 0.001 to 0.1% by weight of Na ₂ HPO ₄ , ONPG mixed solution containing 0.01 to 1% by weight of ONPG and 99 to 99.99% by weight of distilled water is to contain a volume ratio 3: 1.

Beta-galactosidase activity test composition of the present invention can be used by titration to pH 7.0 to 8.0, most preferably 7.3.

The present invention also provides a method for identifying and testing using gelatinase activity. The identification method is to distinguish the microorganisms expressing gelatinase, and since the pH of the reactant is increased when the gelatin is decomposed, it can be confirmed whether or not the gelatinase reaction with the pH indicator. Preferred pH indicators are phenol reds. Phenolic red has a pinkish red color with increasing pH.

The composition for testing gelatinase activity is 0.0001 to 0.1% by weight of cacitone, 0.0001 to 0.1% by weight of yeast extract, 0.01 to 1% by weight of (NH ₄ ) 2HPO ₄ , 0.001 to 0.1% by weight of KCl, 1 to 20 Wt% gelatin, 0.0001-0.1 wt% phenol red and 80-99.99 wt% distilled water, with the pH of the composition being preferably 6.0-7.0. The most preferred pH is 6.5.

In another aspect, the present invention provides an identification method and a test composition using the Esculin hydrolysis (ESC) reaction. The identification method confirms the ability of the microorganism to decompose the esculin, and when decomposed the esculin is combined with Fe ³⁺ ions to confirm that it is brown.

The esculin hydrolysis test composition of the present invention comprises 0.01 to 1% by weight of bacto peptone, 0.01 to 1% by weight of esculin, 0.001 to 0.1% by weight of ferric citrate and 98 to 99.99% by weight of distilled water. .

The present invention also provides an identification method and a test composition using an organic acid alkalizing reaction. The identification method is to check the increasing pH due to the alkalizing reaction of the organic acid when the organic acid is used as a carbon source microorganisms. When BTB is used as a pH indicator, it will turn blue when an alkalization reaction occurs. The organic acid is gluconate, malate, adipate and citrate.

The composition for testing the organic acid alkalizing reaction is 0.001 to 0.1% by weight of MgSO ₄ .7H ₂ O, 0.01 to 1% by weight of NH ₄ H ₂ PO ₄ , 0.01 to 1% by weight of K ₂ HPO ₄ , 0.01 to 1% by weight NaCl, 0.0001 to 0.1 wt% BTB, 0.01 to 5 wt% organic acid and 92 to 99.99 wt% distilled water, with a pH of preferably 5.5 to 6.5. The most preferred pH is 6.0. The organic acid is preferably at least one selected from the group consisting of gluconate, maleate, adipate and citrate, and it is preferable to prepare and use a test composition for each of the four organic acids.

The present invention also provides an identification method and a test composition using the urease activity. The identification method is to confirm whether the microorganism urease expression by confirming the production of ammonia by the urease. When microorganisms express urease, they break down urea to produce ammonia, at which time the pH increases. The pH change can be confirmed by using red cresol red.

The urease activity assay composition comprises 0.01 to 1 wt% of bacto peptone, 0.01 to 1 wt% dextrose, 0.01 to 1 wt% NaCl, 0.01 to 1 wt% KH ₂ PO ₄ , 0.0001 to 0.1 wt% Cresol red chloride of 1 to 20% by weight of urea and 75 to 99% by weight of distilled water, the pH is preferably 6.0 to 7.5, most preferably pH 6.8.

The present invention also provides an identification method and a test composition using the arginine dihydrolase activity. The identification method is to examine the expression of the microorganism arginine dehydrolase by confirming the transformation of amino acids into amines. When a microorganism expresses arginine dihydrolase, it converts amino acids into amines, thereby increasing the pH. The pH change can be confirmed by using red cresol red.

The composition for the test of arginine dehydrolase activity is 0.1 to 10% by weight of bacto peptone, 0.01 to 10% by weight of yeast extract, 0.01 to 1% by weight of dextrose, 0.01 to 1% by weight of agar, 0.01 to 10% by weight. L-arginine, 0.0001 to 1% by weight of cresol red chloride and 74 to 99.99% by weight of distilled water, with a pH of preferably 6.0 to 7.5. The most preferred pH is 6.8.

In another aspect, the present invention provides an identification method and a test composition using a hydrogen sulfate generation reaction. This is a method of distinguishing S availability of microorganisms. When the microorganism separates S from amino acids in the medium, S binds to H to form hydrogen sulfate (H ₂ S), and becomes black.

The composition for testing the hydrogen sulfide production reaction is 1 to 10% by weight of bacto peptone, 0.001 to 1% by weight of paroxy ammonium citrate, 0.001 to 1% by weight of sodium thiosulfate, 0.001 to 1% by weight of methionine, 0.001 to 1 Wt% cysteine, 0.01 to 1 wt% NaCl and 85 to 99.9 wt% distilled water, with a pH of 6.8 to 7.8 being preferred. The most preferred pH is 7.3.

In another aspect, the present invention provides an identification method and a test composition using a fermentation reaction. This is to confirm the fermentation of microorganisms, when the microorganism is a fermentation group to reduce the pH by fermenting glucose, in the case of non-fermentation group does not occur pH change does not occur. Therefore, pH change can be distinguished by fermentation using an indicator such as phenol red. Phenolic red is yellow under acidic conditions.

The fermentation test composition comprises 0.1 to 10% by weight of polypeptone, 0.1 to 10% by weight of dextrose, 0.01 to 1% by weight of K ₂ HPO ₄ , 0.001 to 0.1% by weight of phenol red, 0.01 to 1% by weight of Agar and 78-99.9% by weight of distilled water, the pH of which is preferably 6.5-7.5, most preferably pH 6.9.

The present invention also provides an identification method and a test composition using the nitric acid reduction reaction. This is a way to distinguish the nitric acid reduction of microorganisms, nitric acid is reduced to produce nitrous acid. At this time, the reaction of alpha-naphthylamine and sulfanyl acid is confirmed by the color change to generate a nitric acid reduction reaction. When the nitrate reduction reaction occurs, it becomes dark purple due to the indicator.

The nitric acid reduction test composition includes 0.1 to 10% by weight of nitric acid, 0.01 to 1% by weight of KNO ₃ and 89 to 99% by weight of distilled water. Also included as color indicators of pH changes include alpha-naphthylamine solution and sulfanylinic acid solution. The indicator is added after reacting the microbial sample and the composition for the test for reducing nitrate. The alpha-naphthylamine solution is prepared by mixing 0.1 to 5 g of alpha-naphthylamine per 100 ml of 5N acetic acid, and the sulfanylic acid solution is used to prepare 0.1 to 5 g of sulfanylic acid per 100 ml of 5N acetic acid. Prepare by mixing.

In another aspect, the present invention provides a method for identifying and testing using an indole production reaction. This is a way to determine whether microorganisms break down tryptophan in the medium to produce indole. When indole is produced, the color becomes red so that indole generation can be easily determined.

The indole production test composition comprises 0.1 to 20% by weight of the bacto tryptophan, 0.1 to 10% by weight of L- tryptophan and 70 to 99% by weight of distilled water, the pH is preferably 7.0 to 8.0. In addition, a solution prepared by mixing 50 to 200 ml of isoamyl alcohol, 1 to 20 g of p-dimethylaminobenzaldehyde, and 50 to 200 ml of HCl is used as an indicator.

Thus, each of the above-mentioned identification methods and compositions can be used to identify non-fermented Gram-negative bacteria.

In another aspect, the present invention provides a kit for identifying non-fermentative Gram-negative bacteria comprising at least one composition selected from the group consisting of the above test compositions.

Preferred kits for identification include glucose test composition (GLU), lactose test composition (LAC), sucrose test composition (SUC), mannitol test composition (MAN), maltose test composition (MAL), mannose test Composition (MNN), rhamnose test composition (RHA), galactose test composition (GAL), fructose test composition (FRU), xylose test composition (XYL), arabinose test composition (ARN), beta -Galactosidase test composition (ONPG), gelatinase test composition (GEL), esculin hydrolysis test composition (ESC), gluconate alkylation test composition (GCN), adipate alkylation test composition ( ADI), malate alkylation test composition (MAA), citrate alkylation test composition (CIT), urease test composition (URE), arginine dihydrolase test composition (ADH), hydrogen sulfide test composition ( H ₂ S), composition for fermentation testing (F) , Nitric acid reduction test composition (NIT) and indole production test composition (IND) are included.

The method of preparing the kit for identification is to prepare the above-mentioned compositions by putting the composition per well into a reactor having several wells, and more preferably to dry each composition on the wells.

In the method of using the identification kit, the microorganisms to be identified are first cultured in a plate medium, and then a single colony is diluted in distilled water with turbidity 2, and the diluent is added to the reactor containing the test composition and reacted to each well. Then, the wells containing the urease test composition (URE), the arginine dihydrolase test composition (ADH), the hydrogen sulfide test composition (H ₂ S), and the fermentation test composition (F) cover the upper layer with oil, The reactor is incubated in a 30 ° C. incubator for 24 to 48 hours and then the color change per well is read.

The composition, reaction, and reaction results of the kit for identification of the present invention are briefly shown in Table 1 below.

Composition Inspection Reaction / Reactase result Item - + One GLU Glucose Oxidation Blue-> Light Green yellow 2 LAC Lactose 3 SUC Sucrose 4 MAN Mannitol 5 MAL Maltose 6 MNN Mannose 7 RHA Rhamnos 8 GAL Galactose 9 FRU Fructose 10 XYL Xylose 11 ARN Arabinos 12 ONPG ONPG Beta-galactosides Colorless yellow 13 GEL gelatin Gelatine Yellow-> orange light Red-> pink 14 ESC Esculin Esculin hydrolysis Colorless to light gray Brown-> black 15 GCN Gluconide Alkalizing Yellow-> green blue 16 ADI Adipate 17 MAA Malate 18 CIT Site rate 19 Add oil to upper layer URE Urea Urease Yellow-> orange Red-> pink 20 ADH Arginine Arginine Dihydrolase 21 H ₂ S Sodium sulfate H ₂ S generation Colorless to yellow Gray-> black 22 F Glucose Fermentation Red-> orange yellow 23 NIT Potassium nitrate NO ₂ generation NIT1 + NIT2 / Immediately after addition Light pink without discoloration Purple 24 IND Tryptophan Indole generation IND / 15 seconds later yellow Red belt

After confirming the reaction of the test composition through the color of Table 1, the result is input to the microorganism identification program to read the name of the strain. The microorganism identification program has data in which the reactivity of the test composition of the present invention for each microorganism is input, and if only the reaction is input, the microorganism name is immediately determined. In addition, the microorganism identification program calculates the reactivity of the test composition as a score according to the characteristic biochemical characteristics of each microorganism, thereby easily determining the name of the microorganism by the sum of each score. This is the method used in conventional identification programs.

As an example, the microorganism-specific reactivity for the identification kit of the present invention is shown in Table 2 below as a value for calculating the microorganism identification program.

GLU LAC SUC MAN MAL MNN RHA GAL FRU XYL ARN ONPG GEL ESC GCN ADI MAA CIT URE ADH H ₂ S F NIT IND Achromobacter (Alcaligenes) xylosoxidans ssp xylosoxidans 100 0 0 0 0 0 0 0 0 91 31 0 0 0 39 89 100 100 0 0 0 2 100 0 Acinetobacter baumannii 100 84 2 11 53 99 63 100 2 100 79 0 16 One 20 80 100 62 20 21 0 40 9 0 Acinetobacter haemolyticus 10 10 0 0 38 10 60 10 0 88 50 0 50 0 0 2 99 71 13 0 0 0 0 0 Acinetobacter lwoffii 14 10 0 0 0 14 0 10 0 52 48 0 0 0 3 28 66 21 33 63 0 3 28 0 Alcaligenes faecalis / Alcaligenes sp 30 24 0 0 12 24 12 24 12 36 32 0 0 0 0 24 54 56 18 6 0 12 6 0 Bordetella bronchiseptica 0 0 0 0 0 0 0 0 0 0 0 One 0 0 0 94 85 98 100 60 0 0 40 40 Branhamella catarrhalis 0 0 0 0 0 999 50 999 50 0 50 50 0 0 999 999 999 0 0 50 0 0 92 0 Burkholderia cepacia 90 90 86 100 99 100 0 90 100 100 72 79 90 83 97 93 100 94 60 One 0 10 57 0 Chryseobacterium indologenes 0 0 0 0 0 0 0 0 0 0 0 33 0 78 33 33 33 22 33 0 0 67 0 67 Chryseobacterium meningosepticum 35 35 0 22 13 13 9 0 13 13 13 74 56 100 30 30 26 26 0 5 0 17 0 65 Chryseomonas luteola 100 27 12 100 100 100 86 100 100 100 99 100 61 100 85 One 94 100 64 100 0 0 62 0 Flavimonas oryzihabitans 100 0 25 100 97 100 87 100 100 100 99 10 5 0 99 2 99 97 28 0 0 0 0 0 Moraxella lacunata 0 0 0 0 0 0 0 0 0 0 0 0 42 0 0 0 9 0 0 0 0 0 100 0 Oligella ureolytica 0 0 0 0 0 0 0 0 0 0 0 0 0 0 One One 95 14 100 0 0 0 100 0 Oligella urethralis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 One 99 0 0 0 0 0 0 0 Pseudomonas aeruginosa 97 One 2 90 2 96 22 97 76 90 95 4 82 15 97 98 100 99 57 97 0 2 89 0 Pseudomonas fluorescens 100 24 0 53 33 98 0 100 88 100 100 2 39 0 61 0 79 93 52 80 0 One 22 0 Pseudomonas putida 100 0 0 0 0 100 34 99 80 100 56 2 6 0 97 16 100 94 57 100 0 33 0 0 Pseudomonas stutzeri 100 0 0 60 60 100 0 100 100 100 100 0 0 0 100 60 100 100 33 0 0 0 100 0 Shewanella putrefaciens 0 0 0 0 0 0 0 0 0 67 67 0 11 0 11 11 33 0 0 0 56 0 100 0 Sphingobacterium mμtivorum 100 100 100 0 100 100 0 100 100 100 91 0 0 100 0 0 One 0 100 0 0 0 6 0 Sphingomonas paucimobilis 100 100 100 0 100 100 58 100 100 100 50 100 0 91 34 3 61 50 0 8 0 0 0 0 Stenotrophomonas maltophilia 3 2 2 11 73 2 0 0 5 0 One 83 95 100 7 5 99 87 0 19 0 0 78 0 Weeksella virosa 0 0 0 0 0 0 One 0 0 0 One 0 100 0 One 0 3 0 0 0 0 0 0 100

Strains that can be identified by the non-fermentative Gram-negative bacteria identification composition and kit of the present invention are spleen bacteria, typically Achromobacter ( Alcaligenes ) xylosoxidans ssp xylosoxidans , Acinetobacter baumannii , Acinetobacter haemolyticus , Acinetobacter lwoffii , Alcaligenes sp, Alcaligenes faecalis , Bordetella Bordetella bronchiseptica , Branhamella catarrhalis , Burkholderia cepacia , Chryseobacterium indologenes , Chryseobacterium mendogoceticum Chryseobaterium meningosepticum ), Chryseomonas luteola , Flavimonas oryzihabit ans ), Moraxella lacunata , Oligella ureolytica , Oligella urethralis , Pseudomonas aeruginosa , Pseudomonas aureuginosa , Pseudomonas fluorescens Pseudomonas putida , Pseudomonas stutzeri , Shewanella putrefaciens , Sphingobacterium mμtivorum , Sphingomonas passimobilis ( Sphingomonas paucimobilis ), Stenotrophomonas maltophilia and Weeksella virosa .

The method for identifying the non-fermented Gram-negative bacteria of the present invention can be easily and simply identified by testing on one plate at a time without performing various known identification methods several times. It can be used as an identification kit in hospitals that need to be judged quickly.

The present invention will be described in more detail with reference to the following Examples. The following examples are only for illustrating the present invention, but the protection scope of the present invention is not limited to the following examples.

Example 1 Preparation of Composition for Testing Sugar Availability of Microorganisms

Since microorganisms use different sugars as energy sources, a composition capable of confirming this was prepared. A composition was prepared in which the availability of glucose, lactose, sucrose, mannitol, maltose, mannose, rhamnose, galactose, fructose, xylose and arabinose was confirmed, and the microorganisms were reacted with the composition to color the composition. It is designed to confirm the reactivity with the change.

When the microorganism uses a specific sugar as an energy source, the sugar in the composition is oxidized to yellow, and when not used, it is blue.

1-1. Trace Element Stock Production

0.09 g of sodium molybdate (Na ₂ MoO ₄ · 2H ₂ O, Duksan), 0.03 g of boric acid (H ₃ BO ₃ , Duksan), 0.02 g of cobalt chloride (CoCl ₂ · 6H ₂ O, Duksan), zinc sulfate (ZnSO ₄ · 7H ₂ O, Duksan) 0.01 g, 0.3 g / ㎖ manganese chloride (MnCl ₂ · 4H ₂ O, Duksan) 0.001 ㎖, 0.3 g / ㎖ sodium selenite (Na ₂ SeO _3, Duksan) 0.001 ㎖, 0.2 g A total of 100 ml of trace urea stock solution was prepared by mixing 0.001 ml / ml nickel chloride (NiCl ₂ · 6H ₂ O), 0.001 ml of 0.1 g / ml cupric chloride (CuCl ₂ · 2H ₂ O, Deoksan) and distilled water. It was refrigerated.

1-2. Vitamin Stock Production

0.0001 g of vitamin B12, 0.002 g of biotin (sigma Cat No. B-4501), 0.002 g of polylic acid (sigma Cat No. F-7876), 0.005 g of riboflavin (sigma Cat No. R-4500), nicotinic acid (sigma Cat No N-33761) 0.005 g, lipoic acid (sigma Cat No. T-5625) 0.005 g, ρ-aminobenzoic acid (sigma Cat No. A-9878) 0.005 g, thiamine (sigma Cat No. T-4625) 0.005 g, calcium DL-pantothenate (sigma Cat No. P-2250) and 0.01 g pyridoxine (sigma Cat No. P-9755) were mixed in tertiary distilled water to prepare a total 1000 ml of vitamin stock solution. The vitamin stock solution was refrigerated while avoiding light contact with foil.

1-3. Oxidation Media Preparation

NH ₄ H ₂ PO ₄ (monobasic, Deoksan) 0.3 g, MgSO ₄ · 7H ₂ 0 (showa) 0.06 g, KCl (Deoksan) 0.16 g, yeast extract (difco Cat No.212750) 0.033 g, casamino acid (difco Cat No.223050) 0.033 g, polypeptone (hispan Cat No. 1610.01) 0.066 g, soy peptone (difco Cat No. 211677), 0.033 g of trace urea stock solution and 3 ml of tertiary distilled water were mixed to prepare 100 ml and then boiled. After cooling at room temperature. Thereafter, 0.6 ml of 0.0005 g / ml CaCl ₂ (Duksan) was added, and then adjusted to pH 7.6, and when the solution became cloudy, it boiled. Here, 0.3 ml of vitamin stock solution and ferric ammonium citrate (0.05 g / ml, 0.6 ml of sigma F-5879) and 16 ml of BTB (0.001 g / ml 20% ethanol, showa 0221-5130) were mixed, and then the pH was adjusted to 7.6 again.

1-4. Preparation of composition for testing sugar availability of microorganisms

Glucose (Cat No. 215530, difco), Lactose (Cat No. 215620, difco), Sucrose (Cat No. S7903, sigma), Mannitol (Cat No. 0170-17-5, difco), Maltose (Cat No M-5884, sigma, Mannose (Cat No. M-8296, sigma), Rhamnose (Cat No. R-3875, sigma), Galactose (Cat No. G-6404, sigma), Fructose (Cat No. 6 g each of F-0127, sigma), xylose (Cat No. X-1500, sigma) and arabinose (Cat No. A-3256, sigma) were added to 100 ml of oxidation medium, mixed well and pH Was adjusted to 7.6. After filtration with 0.2 um filter paper and stored in a cool dark place.

1-5. Accuracy verification of microbial sugar availability test compositions

The accuracy of the standard microorganisms received from the ATCC using the composition prepared in 1-4 was verified by verifying the accuracy.

50 μl of the sugar-availability test composition of 1-4 was put in a well plate and dried in a 25 ° C. dryer.

The microorganisms were cultured in a plate medium and then a single colony was taken to prepare a dilution diluted in distilled water with turbidity 2.

Diluted solution was added to each well containing the composition for testing sugar availability and incubated at 30 ° C. for 24 hours. After incubation, the color of each well was checked by changing the color of yellow (+) and blue (-using sugar).

microbe Glucose Lactose Sucrose Mannitol Maltose Mannose Lanos Galactose Fructose Xylose Arabinos Pseudomonas aeruginosa ATCC 9027 + - - + - + - + + + + Aximetobacter Baumani ATCC 19606 + + - - ± + + + - + + Stenotropomonas Maltopila ATCC 51331 - - - - - - - - - - - Burgunderia Sephacia ATCC 25608 + + + + + + - + + + + Pseudomonas Fluorescein ATCC49838 + - - + - + - + + + + Alkali Genes Packalis ATCC 8750 - - - - - - - - - - - Axinetobacter Haemolyticus ATCC19002 - - - - - - - - - - - Cryeobacterium Mening Oceptime ATCC13253 + + - + + + - - + - - Axinetobacter Rupee ATCC15309 - - - - - - - - - - - Cryeobacterium indologenes ATCC49471 - - - - - - - - - - - Pseudomonas Stuzeri ATCC17588 + - - + + + - + + + + Pseudomonas putida ATCC49128 + - - - - + - + + + +

The results confirmed in Table 3 were the same as the sugar availability of the standard microorganisms.

1-6. Identification Results According to pH Change of Sugar Solubility Testing Composition

It was titrated to the water pH 6.7 which prepared the sugar availability test composition by the method of 1-4. Then, the reactivity was tested for 20 strains (Table 4).

microbe Glucose Sucrose Mannitol Maltose Standard result Experiment result Standard result Experiment result Standard result Experiment result Standard result Experiment result A. baumanniiATCC 19606 - + - - - - - - B. cepacia ATCC 25416 + + + + + + + + C.meningsepticum ATCC 13253 + ± - - + ± + ± C.meningsepticum ATCC 49470 + ± - - + ± - - C. indologenes ATCC 49471 + ± - - - - + ± P. putida ATCC 49128 + + - - - - - - P. stutzeri ATCC 17588 + - - - - - - - P. fluorescens ATCC 49838 + + - - + - - - P. aeruginosa ATCC 9027 + + - - + + - - S. maltophilia ATCC 13637 - - - - - - + - S. maltophilia ATCC 51331 - - - - - - + ± S. putrefaciens ATCC 49138 - - - - - - - - P. aeruginosa ATCC 27853 + + - - + + - - B. bronchiseptica ATCC 10580 - - - - - - - - B. bronchiseptica ATCC 4617 - - - - - - - - B. cepacia ATCC 25608 + + + + + + + + A. haemolyticus ATCC 19002 - - - - - - - - A. lowffii ATCC 15309 - - - - - - - - Al. faecalisATCC 35655 - - - - - - - - R. picketti ATCC 49129 - + - - - - - ±

In Table 4, the standard result is a result known from the biochemical characterization of each strain, and the experimental result is a result of the composition of pH 6.7. The sugar availability test composition was unable to confirm the exact result at pH 6.7, and pH 7.6 was optimal.

Example 2: Preparation of a composition for beta-galactosidase activity test

A composition for confirming the beta-galactosidase activity of a microorganism, wherein the microorganism decomposes ONPG when it expresses beta-galactosidase, and yellow when it does not express beta-galactosidase. To prepare a composition exhibiting no color change.

2-1. 1% peptone medium

0.9 g of bacto peptone (difco 211677) was mixed with tertiary distilled water to prepare 100 ml, heated and cooled at room temperature. The peptone medium was then adjusted to pH 7.3 and sterilized at 121 ° C. for 15 minutes.

2-2. ONPG Solution

0.042 g of Na ₂ HPO ₄ (showa) was dissolved in tertiary distilled water to prepare a 100 ml mixture, which was then adjusted to pH 7.3 and cooled. Then, ONPG solution was prepared by adding 0.18 g of ONPG (sigma N-1127) to the mixed solution in a cold dark place and storing it in a frozen state.

2-3. Beta-galactosidase activity test composition

The 1% peptone medium and ONPG solution prepared above were mixed in a 3: 1 volume ratio, filtered through a 0.2 um filter, and then stored in a cool dark place.

2-4. Accuracy Verification of Compositions for Testing Beta-galactosidase Activity

The experiment was carried out in the same manner as in Example 1-5, and the results are shown in Table 5 below.

microbe ONPG reaction Pseudomonas aeruginosa ATCC 9027 - Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 + Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 + Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 - Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 -

As a result of the experiment, each standard microorganism showed the same reactivity with the expression of beta-galactosidase.

Example 3: Composition for Gelatinase Activity Test

A composition was prepared to confirm whether the microorganism dissolved gelatin. When the microorganism dissolves the gelatin, the pH of the composition containing the gelatin increases, so that the color of the indicator in the composition becomes pink red. If the microorganism is unable to dissolve gelatin, the composition for testing gelatinase activity is yellow.

3-1. Composition preparation

0.033 g of citaton (difco 0259-17), 0.033 g of yeast extract (difco 212750), 0.4 g of (NH4) 2HPO4 (Duksan), 0.08 g of KCl (Duksan), 8 g of gelatin (difco 0143-17), 0.045 g / 100 μl of a phenol red (sigma P-4758) was mixed with distilled water in tertiary distilled water to prepare 100 mL of the GEL test composition. After heating, the mixture was cooled to room temperature, adjusted to pH 6.5, sterilized, and stored at room temperature.

3-2. Validation of the composition's accuracy

The experiment was carried out in the same manner as in Example 1-5, and the results are shown in Table 6 below.

microbe Gelatin reaction Pseudomonas aeruginosa ATCC 9027 + Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 + Burgunderia Sephacia ATCC 25608 + Pseudomonas fluorescein ATCC 49838 + Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 + Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 - Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 +

As a result of the experiment, each standard microorganism showed reactivity with the composition for testing gelatinase activity in the same manner as gelatin dissolution.

Example 4: Preparation of the composition for the test of esculin hydrolysis reaction

A composition for measuring the esculin hydrolysis of microorganisms was prepared. When microorganisms break down esculin in the composition, they bind with Fe ³⁺ ions in the composition, resulting in brown color. Colorless if not decomposed.

4-1. Preparation of the composition for the test of esculin hydrolysis reaction

0.5 g of bactopeptone (difco 211677), 0.4 g of esculin (sigma E-8250) and 0.05 g of ferric citrate (sigma F-6129) were mixed in distilled water to prepare 100 ml of the ESC test composition. Cooled at and then sterilized and refrigerated.

4-2. Accuracy Verification of Compositions for the Test of Esculin Hydrolysis

The experiment was carried out in the same manner as in Example 1-5, and the results are shown in Table 7 below.

microbe Eskuline reaction Pseudomonas aeruginosa ATCC 9027 - Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 + Burgunderia Sephacia ATCC 25608 + Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 + Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 + Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 -

The results shown in Table 7 were the same as whether the standard microorganisms were degraded esculin.

Example 5 Composition for Examining Organic Acid Alkalization Reaction

A composition for measuring whether the microorganism uses each organic acid as a carbon source during metabolism was prepared.

When a microorganism uses an organic acid as a carbon source, the composition is alkylated to change the color of the composition to blue, and when not available, yellow without color change.

5-1. Preparation of gluconate test composition

0.02 g MgSO ₄ 7H ₂ O (shinyo), 0.1 g NH ₄ H ₂ PO ₄ (monobasic, shinyo), 0.1 g K ₂ HPO ₄ , 0.5 g NaCl and BTB (BromThymol Blue sodium salt, sigma B-8630) 0.024 g was mixed with distilled water to a total of 100 ml, heated and cooled to adjust the pH to 6.0. Thereafter, 0.6 g of gluconate ( _d -Gluconic acid (potassium salt), sigma G-4500) was mixed, and the pH was adjusted to 6.0 again, and then stored in a dark cool place after sterilization.

5-2. Preparation of maleate test composition

0.02 g MgSO ₄ 7H ₂ O (shinyo), 0.1 g NH ₄ H ₂ PO ₄ (monobasic, shinyo), 0.1 g K ₂ HPO ₄ , 0.5 g NaCl and BTB (BromThymol Blue sodium salt, sigma B-8630) 0.024 g was mixed with distilled water to a total of 100 ml, heated and cooled to adjust the pH to 6.0. Thereafter, 1.2 g of malate ( _DL -Malic acid (disodium salt), sigma M-6773) was mixed to adjust the pH to 6.0 again, and then stored in a dark cool place after sterilization.

5-3. Preparation of adipate test composition

0.02 g MgSO ₄ 7H ₂ O (shinyo), 0.1 g NH ₄ H ₂ PO ₄ (monobasic, shinyo), 0.1 g K ₂ HPO ₄ , 0.5 g NaCl and BTB (BromThymol Blue sodium salt, sigma B-8630) 0.024 g was mixed with distilled water to a total of 100 ml, heated and cooled to adjust the pH to 6.0. Thereafter, 0.6 g of adipate (Adipic acid, sigma A-5252) was mixed and adjusted to pH 6.0 again, and then stored in a dark cool after sterilization.

5-4. Preparation of citrate test composition

0.02 g MgSO ₄ 7H ₂ O (shinyo), 0.1 g NH ₄ H ₂ PO ₄ (monobasic, shinyo), 0.1 g K ₂ HPO ₄ , 0.5 g NaCl and BTB (BromThymol Blue sodium salt, sigma B-8630) 0.024 g was mixed with distilled water to a total of 100 ml, heated and cooled to adjust the pH to 6.0. Since the citrate (Sodium citrate, sigma S-4641) 0.6 g was mixed again to adjust the pH to 6.0, and then sterilized and stored in a dark cool.

5-5. Accuracy Verification of Composition for Testing Organic Acid Alkali Reaction

The experiment was carried out in the same manner as in Example 1-5, and the results are shown in Table 8 below.

microbe Gluconate Adipate Malate Site rate Pseudomonas aeruginosa ATCC 9027 + + + + Axinetobacter Baumani ATCC 19606 - + + + Stenotropomonas Maltopila ATCC 5133 + + + + Burgunderia Sephacia ATCC 25608 + + + + Pseudomonas fluorescein ATCC 49838 + - + + Alkali Genes Packalis ATCC 8750 - - + + Axinetobacter Haemolyticus ATCC 19002 - - + + Cryeobacterium Meningopceptum ATCC 13253 - - - - Axinetobacter Rupee ATCC 15309 - - - - Cryeobacterium Indologenes ATCC 49471 - - - - Pseudomonas Stuzeri ATCC 17588 + - + + Pseudomonas Putida ATCC 49128 + - + +

As a result of testing the standard microorganism reactivity with the organic acid alkalizing reaction test composition of the present invention, it showed the same reactivity as the organic acid availability of each standard microorganism.

Example 6: Composition for Testing Urease Activity

A composition was prepared to determine whether the microorganism contains a urease capable of producing ammonia from urea.

The composition for the urease activity test produces ammonia by decomposing urea in the presence of urease, thereby increasing the pH of the composition and making the composition red. In the absence of reaction, the composition turned yellow without color change.

6-1. Preparation of urease activity test composition

0.1 g bactopeptone (difco 211677), 0.1 g dextrose (difco 215530), 0.5 g NaCl, 0.2 g KH ₂ PO ₄ (shinyo) and 0.015 g Cresol red sodium salt (aldrich 11,448-0) The mixture was well mixed with 80 mL of distilled water and then sterilized by adjusting to pH 6.8. 20 ml of urea solution (12 g of urea (sigma, U-0631), distilled water) mixed with a sterilized 80 ml solution at 0.2 um were mixed and stored in a cool dark place.

6-2. Validation of the composition for testing urease activity

After adding the microbial sample to each well was tested in the same manner as in Example 1-5 except that the upper layer was covered with mineral oil, the results are shown in Table 9 below.

microbe Urease reaction Pseudomonas aeruginosa ATCC 9027 + Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 - Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 - Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 + Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 +

As a result of comparing the reaction by the urease of Table 9 with the inclusion of the urease of the standard microorganism, it was confirmed that the same reactivity was shown depending on whether the urease was included.

Example 7 Composition for Testing Arginine Dihydrolase Activity

Arginine dihydrolase is an enzyme that transforms amino acids into amine forms.

When the microorganism expresses arginine dehydrolase, the composition of the test for arginine dehydrolase was prepared such that the pH of the composition was increased while changing the amino acid in the composition to an amine, thereby changing the composition to a red color.

7-1. Preparation of Arginine Dihydrolase Activity Test Composition

1.5 g of bacto peptone (difco 211677), 0.9 g of yeast extract (difco 212750), 0.3 g of dextrose (difco 215530), 0.1 g of agar (difco 214010), 1.5 g of L-arginine (free acid, sigma), cresol red A total of 100 ml of the composition was prepared by mixing 0.016 g of chloride (aldrich 11,448-0) in tertiary distilled water, adjusted to pH6.8, and then sterilized by refrigeration.

7-2. Accuracy Verification of Compositions for Testing Arginine Dihydrolase Activity

The experiment was carried out in the same manner as in Example 6-2, and the results are shown in Table 10 below.

microbe Arginine Dihydrolase Reaction Pseudomonas aeruginosa ATCC 9027 + Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 - Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 + Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 + Cryeobacterium Meningopceptum ATCC 13253 - Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 - Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 +

In Table 10, the standard microorganism arginine dehydrolase reaction was the same as the expression of arginine dehydrase.

Example 8: Composition for testing hydrogen sulfide (H ₂ S) production reaction

In order to measure the ability of microorganisms to separate and absorb S from extracellular amino acids, a composition was prepared in which isolated S was combined with H to appear black.

8-1. Preparation of hydrogen sulfide generation reaction composition

12.96 g of bactopeptone (difco 211677), 0.18 g of parric ammonium citrate (sigma F-5879), 0.18 g of sodium thiosulfate (sigma S-1648), 0.15 g of L-methion (sigma M-6039), L-cysteine (sigma C-4820) 0.15 g and 0.3 g of NaCl were dissolved in tertiary distilled water to prepare a 100 ml composition, and then the pH was adjusted to 7.3. It was then sterilized and stored in a cool dark place.

8-2. Accuracy Verification of Composition for Testing Hydrogen Sulfide Production

The experiment was carried out in the same manner as in Example 6-2, and the results are shown in Table 11 below.

microbe Hydrogen sulfide reaction Pseudomonas aeruginosa ATCC 9027 - Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 - Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 - Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 - Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 -

In Table 11, all of the standard microorganisms used did not use hydrogen sulfide generation reactions.

Example 9 Composition for Fermentation Testing

In order to confirm the fermentation under anaerobic conditions of the microorganisms, a composition was prepared in which the pH decreased during fermentation by color change.

When fermentation occurs under anaerobic conditions, the composition is yellow, and if not, it remains red.

9-1. Preparation of Fermentation Testing Composition

3.15 g polypeptone (hispan 1610.01), 1.5 g dextrose (difco 215530), 0.1875 g K ₂ HPO ₄ , 2 ml phenol red (sigma P-4758, 0.045 g / ml) and 0.12 g agar (difco 214010) A total of 100 ml of the composition was prepared by mixing in tertiary distilled water, mixed well and adjusted to pH 6.9. After sterilization was refrigerated.

9-2. Validation of the composition for fermentation testing

The experiment was carried out in the same manner as in Example 6-2, and the results are shown in Table 12 below.

microbe Fermentation reaction Pseudomonas aeruginosa ATCC 9027 - Axinetobacter Baumani ATCC 19606 + Stenotropomonas Maltopila ATCC 51331 - Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 - Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 - Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 -

In Table 12, only Axinebacter Baumani fermented under anaerobic conditions among the standard microorganisms used changed the color of the composition to yellow, and the other microorganisms did not show fermentation reaction.

Example 10 Composition for Testing Nitrate Reduction

A composition was prepared to determine the ability of a microorganism to reduce nitric acid to nitrous acid. When the microorganisms reduce the nitric acid, the color of the composition is dark purple, and if no reduction reaction occurs, there is no color change.

10-1. Preparation of nitric acid reduction test composition

5.4 g of nitric acid (difco 0268-17) and 0.4 g of KNO ₃ (Duksan) were mixed with tertiary distilled water to prepare 100 ml of the composition, which was sterilized and refrigerated.

In addition, 100 ml of alpha-naphthylamine solution containing 0.5 g of alpha-naphthylamine and 5 N acetic acid was added to 100 ml of 0.8 g of sulfanic acid and 5 N acetic acid. Ml of sulfanylinic acid solution was prepared respectively.

10-2. Validation of the composition for the test of nitric acid reduction reaction

The experiment was carried out in the same manner as in Example 1-5, and then 50 μl of the alpha-naphthylamine solution and the sulfanylic acid solution were added, and the color change is described in Table 13 below. As a result, the nitrate reduction test composition showed a result consistent with the characteristics of each strain.

microbe Nitrate reduction Pseudomonas aeruginosa ATCC 9027 + Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 ± Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 - Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 + Pseudomonas Stuzeri ATCC 17588 ± Pseudomonas Putida ATCC 49128 -

Example 11 Indole Formation Test Composition

An indole production test composition capable of determining whether or not microorganisms decompose tryptophan in a medium to produce indole was prepared. P-dimethylaminobenzaldehyde was used as an indicator to confirm the indole generation of the composition, a red ring is formed when indole is produced, and color change does not appear when indole is not produced.

11-1. Preparation of indole production reaction composition

A total of 100 ml of the composition was prepared by mixing 6 g of bacto trypan (difco 0123-17-3), 1 g of L-tryptophan (sigma T-0254), and tertiary distilled water, followed by sterilization by adjusting the pH to 7.4.

In addition, 150 ml of isoamyl alcohol, 10 g of p-dimethylaminobenzaldehyde, and 150 ml of HCL were prepared as an indicator. The indicator is cultured in the indole test composition for microorganisms and put 50 ㎕ to observe the color change. When there is no reaction, it shows yellow without color change, and when there is reaction, it shows red color.

11-2. Accuracy Verification of Composition for Testing Indole Production

The experiment was carried out in the same manner as in Example 1-5, after which the indicator was added and the color change is described in Table 14 below.

microbe Indol generation Pseudomonas aeruginosa ATCC 9027 - Axinetobacter Baumani ATCC 19606 - Stenotropomonas Maltopila ATCC 51331 - Burgunderia Sephacia ATCC 25608 - Pseudomonas fluorescein ATCC 49838 - Alkali Genes Packalis ATCC 8750 - Axinetobacter Haemolyticus ATCC 19002 - Cryeobacterium Meningopceptum ATCC 13253 + Axinetobacter Rupee ATCC 15309 - Cryeobacterium Indologenes ATCC 49471 + Pseudomonas Stuzeri ATCC 17588 - Pseudomonas Putida ATCC 49128 -

Example 12 Preparation of a Kit for Identification of Non Fermented Gram Negative Bacteria

A total of 24 compositions prepared in Examples 1 to 11 were placed in 50 μl of each well of a 24-well microplate and dried at 25 ° C. to prepare a kit.

Example 13: Identification of Microorganisms Using a Identification Kit

Three microorganisms (Pseudomonas aeruginosa, Axinetobacter Baumani, Stenotropomonas maltophilia) received from ATCC were each incubated in a plate medium, diluted in distilled water with turbidity 2, and then, this was It was reacted to the prepared kit for identification. Checking the color change according to the composition for each test to determine whether the reaction is shown in Table 15, which was entered into the program for identifying microorganisms. As a result, the inoculated microorganisms were identified as Pseudomonas aeruginosa, Axinetobacter Baumani, and Stenotropomonas maltophilia, respectively.

GLU LAC SUC MAN MAL MNN RHA GAL FRU XYL ARN ONPG Pseudomonas aeruginosa + - - + - + - + + + + - Axinetobacter Baumani + + - - - + + + - + + - Stenotropomonas Maltophilia - - - - - - - - - - - + GEL ESC GCN ADI MAA CIT URE ADH H ₂ S F NIT IND Pseudomonas aeruginosa - - + + + + + + - - + - Axinetobacter Baumani - - - + + + - - - + - - Stenotropomonas Maltophilia + + - - + + - - - - + -

Example 14 Identification of Microorganisms Using a Identification Kit

The identification results of various microorganisms were confirmed using the kit for identification prepared in Example 12, which was compared with commercially available VITEK (bioMeriux) and API 20NE (bioMeriux).

A total of 114 strains (ATCC) were identified between March and April 2002. Identification experiment using VITEK at Seoul National University, identification experiment using the kit of API 20NE and Example 12 was carried out at the Combed Life Science Research Institute, the results analysis is shown in Table 16 by the Combed Life Science Research Institute.

microbe No. (%) of isolates Bacteria Identified as: Species identified Number of bacteria identified with generic name Unidentified bacteria VITEK API Example 12 VITEK API Example 12 VITEK API Example 12 P. aeruginosa 31 31 30 31 One A. baumannii 22 22 22 22 S. maltophilia 11 11 11 11 B. cepacia 12 12 12 12 P. fluorescens 4 3 2 3 One 2 One P. putida 8 4 8 7 4 One A. faecalis 2 2 One One One One C. meningosepticum 4 4 2 2 One 2 One A. xylosoxydans 14 14 12 14 2 A. lowffii 6 6 0 6 4 2 gun 76 76 75 76 0 0 0 0 One 0 Identification accuracy (%) 95.6 87.7 95.6

In Table 16, the identification kit of the present invention shows an accuracy of about 95.6%, which is superior to or similar to the existing identification kit, and can be used to replace the existing identification kit.

As mentioned above, the non-fermentative Gram-negative bacteria identification method of the present invention can not only identify the spleen bacteria by simply examining a total of 24 items, but also has an excellent identification rate for the purpose of diagnosing diseases caused by bacteria. Can be used as

Claims (17)

(a) preparing a suspension of the microorganism to be identified; (b) sugar oxidation, beta-galactosidase activity, gelatinase activity, esculin hydrolysis reaction, organic acid alkalizing reaction, urease activity, arginine dehydrolase activity, hydrogen sulfate formation reaction, fermentation, nitric acid Reacting the suspension with a test composition capable of identifying a reduction reaction and an indole production reaction, respectively; (c) analyzing the reaction results Microbial identification method, including that. The method of claim 1, wherein the sugar is at least one selected from the group consisting of glucose, lactose, sucrose, mannitol, maltose, mannose, rhamnose galactose, fructose, xylose and arabinose. The method of claim 1, wherein the alkalizing reaction is an alkalizing reaction for at least one organic acid selected from the group consisting of gluconate, malate, adipate, and citrate. NH ₄ H ₂ PO ₄ , MgSO ₄ · 7H ₂ 0, KCl, yeast extract, casamino acid, polypeptone, soypeptone, trace urea stock solution, CaCl ₂ , vitamin stock, ferric ammonium citrate, BTB, sugar Including; The trace urea stock solution comprises sodium molybdate, boric acid, cobalt chloride, zinc sulfate, manganese chloride, sodium selenite, nickel chloride and cupric chloride; The vitamin stock solution comprises vitamin B12, biotin, folic acid, riboflavin, nicotinic acid, lipoic acid, p-aminobenzoic acid, thiamine, calcium DL-pantothenate and pyridoxine; Composition for testing sugar availability of microorganisms. (a) a peptone medium comprising 0.1 to 10% by weight of bacto peptone and 90 to 99% by weight of distilled water; And (b) a mixed solution comprising 0.001 to 0.1 wt% Na ₂ HPO ₄ , 0.01 to 1 wt% ONPG and 99 to 99.99 wt% distilled water; Beta-galactosidase activity test composition of the microorganism containing the volume ratio 3: 1 0.0001 to 0.1% by weight of cacitone, 0.0001 to 0.1% by weight of yeast extract, 0.01 to 1% by weight of (NH ₄ ) ₂ HPO ₄ , 0.001 to 0.1% by weight of KCl, 1 to 20% by weight of gelatin, 0.0001 to A composition for testing gelatinase activity of microorganisms comprising 0.1 wt% phenol red and 80 to 99.99 wt% distilled water. A composition for testing an esculin hydrolysis reaction of a microorganism comprising 0.01 to 1% by weight of bacto peptone, 0.01 to 1% by weight of esculin, 0.001 to 0.1% by weight of ferric citrate, and 98 to 99.99% by weight of distilled water. 0.001 to 0.1 wt% MgSO ₄ .7H ₂ O, 0.01 to 1 wt% NH ₄ H ₂ PO ₄ , 0.01 to 1 wt% K ₂ HPO ₄ , 0.01 to 1 wt% NaCl, 0.0001 to 0.1 wt% BTB, 0.01 to 5% by weight of the organic acid and 92 to 99.99% by weight of distilled water comprising a composition for testing the organic acid alkylation reaction of the microorganism. 0.01 to 1 wt% bacto peptone, 0.01 to 1 wt% dextrose, 0.01 to 1 wt% NaCl, 0.01 to 1 wt% KH ₂ PO ₄ , 0.0001 to 0.1 wt% cresol red chloride, 1 A composition for testing urease activity of a microorganism comprising 20 to 20 wt% urea and 75 to 99 wt% distilled water. 0.1 to 10% by weight of bacto peptone, 0.01 to 10% by weight yeast extract, 0.01 to 1% by weight dextrose, 0.01 to 1% by weight agar, 0.01 to 10% by weight L-arginine, 0.0001 to 1 weight A composition for testing the arginine dehydrolase activity of a microorganism comprising% cresol red chloride and 74 to 99.99% by weight distilled water. 1 to 10% by weight of bacto peptone, 0.001 to 1% by weight of paroxy ammonium citrate, 0.001 to 1% by weight of sodium thiosulfate, 0.001 to 1% by weight of methion, 0.001 to 1% by weight of cysteine, 0.01 to 1 A composition for testing a hydrogen sulfide generation reaction of a microorganism comprising wt% NaCl and 85 to 99.9 wt% distilled water. 0.1 to 10 weight percent polypeptone, 0.1 to 10 weight percent dextrose, 0.01 to 1 weight percent K ₂ HPO ₄ , 0.001 to 0.1 weight percent phenol red, 0.01 to 1 weight percent agar and 78 to 99.9 Composition for the test of fermentation of microorganisms comprising distilled water by weight. (a) a mixture comprising 0.1 to 10 wt% nitric acid, 0.01 to 1 wt% KNO ₃ and 89 to 99 wt% distilled water; And (b) alpha-naphthylamine and sulfanylinic acid as indicators; Composition for testing the nitric acid reduction reaction of a microorganism comprising a. (a) a mixture comprising 0.1 to 20% by weight of a bacto tryptophan, 0.1 to 10% by weight of L-tryptophan and 70 to 99% by weight of distilled water; And (b) p-dimethylaminobenzaldehyde as an indicator Indole production reaction test composition of the microorganism comprising a. A microorganism identification kit comprising at least one composition selected from the group consisting of the composition according to any one of claims 4 to 14. 16. The identification kit according to claim 15, wherein the microorganism identification kit identifies non-fermentative Gram-negative bacteria. The kit of claim 15, wherein the composition is a dry matter.