DE2845838A1 - Identification of microorganisms, esp. Neisseria spp. - by colour reaction with specific substrate and dye - Google Patents

Abstract

Identification is carried out by (a) adding a predetermined amt. of a saline contg. the microorganism to a soln. contg. a diazonium dye detector and a substrate, (b) incubating in an aq. meidum with a buffer at 35 degrees C. for 5 hrs., and (c) detecting a positive or negative colour reaction. Specifically, the substrate is selected from beta-naphthyl-beta-D-galactopyranoside (I), N-L-glutamyl-beta-naphthyl-amide (II), L-hydroxyproline-beta-naphthylamide (III), L-serine-beta-naphthylamide (IV), L-arginine-beta-naphthylamide (V), glycine-glycine-beta-naphthylamide (VI), beta-naphthylphosphate (VII), beta-naphthyl valerate (VIII), 4-methoxy-leucine-beta-naphthylamide (IXe and glycine-beta-naphthylamide (X). Method is esp. useful for distinguishing between N. gonorrhoeae, N. meningitidis and N. lactamica. The identification can be made rapidly (e.g. 1-4 hr.) by comparison with a standard chart.

Description

Method and device for rapid

Identification of microorganisms, especially Neisseria gonorrhoeae and Neisseria meningitidis The invention relates to a diagnostic Microbe identification system. In particular, it relates to a method for Identification of Neisseria species, particularly N. gonorrhoeae and N. meningitidis. It also relates to the reagents for use in the method and an apparatus for carrying out the method, consisting of a test strip with a number small container or reaction vessel, each a synthetic substrate and contain a buffer and take up a test inoculum during incubation can.

The incidence of gonorrhea has reached epidemic proportions.

In 1975 there were approximately three million in the United States Cases of gonorrhea noted. A system for the rapid identification of N. gonorrhoeae is an essential aid in identifying the infection and the following treatment of the patient. The term gonorrhea includes all infections caused by the gonococcus Neisseria gonorrhoeae will.

The isolation of N. gonorrhoeae from Non-genital areas as well as the isolation of N. meningitidis and N. lactamica from the genital area poses a problem in the clinical laboratory. All of these Species of Neisseria grow on those used to isolate N. gonorrhoeae Media. Therefore, the isolates have to be further characterized in order to determine their identity of the organisms.

The usual techniques for identifying N. gonorrhoeae require the cultivation of a sample on a selective medium, followed by an oxidase test, a Gram stain and an examination of the morphology for a pre-identification from N. gonorrhoeae carry out. Confirmation of identification according to the usual Working methods also require an incubation on a non-selective ven Enrichment medium, such as a chocolate agar plate, to ensure a sufficient Get growth for the confirmatory testing.

An 18- to 24-hour incubation is required to achieve adequate To maintain growth. The growth from the previous medium is on a Carbohydrate degradation media are inoculated, usually from a series of the following Carbohydrates: glucose (dextrose), maltose, sucrose, fructose, levulose and lactose sugar in cystine trypticase agar base with phenol red indicator. One incubates under aerobic conditions to break down the carbohydrate for up to two days as needed. During this period the carbohydrates can be converted into acidic end products will. The presence of acid is indicated by the change in the color of an indicator, like phenol red, determined from red to yellow. This will get a positive response displayed. The usual technique for performing the confirmatory examination can be require up to three days.

There are also some ways to work for faster identification from N. gonorrhoeae. The known rapid methods use an initial isolation on a selective medium, as well as the usual technique, followed by inoculation on a primary isolation plate to grow the inoculum. Then will inoculated the carbohydrate breakdown media with a very dense suspension of the bacteria and incubated in a water bath for up to 5 hours to produce an acidic reaction to evoke. In other words, a small amount of substrate is used and a very dense bacterial suspension and accelerates the reaction. This work Under these conditions, wise are not of the growth of the organism, but of the growth of the organism depends on the presence of preformed enzyme of the various substrates degrades.

The modified Thayer-Martin (MTM) medium is generally recognized to isolate the gonococcus and meningococcus from places where these organisms are far exceeded in number by natural bacterial flora (J.D. Thayer and J.E. Martin Je. : Improved Medium Selective for the Cultivation of N. gonorrhoeae and N. meningitidis. Public Health Rep. 81: 559-562, June 1966).

In T-M medium, the overgrowth becomes more gram-positive and more gram-negative Preventing bacteria by adding vancomycin to gram-positive contaminants to inhibit sodium colistimate for the gram-negative flora and nystatin to To inhibit yeasts which are sometimes troublesome in vaginal and rectal cultures. Since the MTM medium contains antimicrobial agents, bacteria will grow and Fungi and Neisseria with the exception of N. gonorrhoeae, N. meningitidis and N. lactamica, suppressed.

Specimens suspected of having a gonococcus (GC) or. Meningococcus are placed on an MTM medium and soaked in carbon dioxide incubated at 350 for three days.

Isolates are selected from the growth for positive identification. However, the identification of organisms after isolation is on the MTM medium laborious and time consuming. Cleaning requires 24 hours followed by additional 5 to 48 hours for the identification So requires the identification (after the Isolation from the MTMX for at least approximately 30 hours and sometimes four days, depending on the intricacies of the organism and the type of identification tests used.

After isolation on the MTM medium, an enrichment medium, typically a chocolate agar plate, inoculated and incubated for 18 to 24 hours, to obtain sufficient inoculum growth for confirmatory testing. As soon sufficient growth is available, an inoculum of the Growth produced in water and used to inoculate a carbohydrate breakdown medium, which usually consists of a range of glucose, sucrose, fructose and similar sugars, consists. The purpose of breaking down carbohydrates is to make up glucose and others Sugars produce acid when taking the test in cystine trypticase agar (CTA) medium performs.

A faster system for identifying N. gonorrhoeae was made Developed by Kellogg and Turner (D.S. Kellogg Jr. and E.M. Turner, 1973, Rapid Fermentation Confirmation of Neisseria Gonorrhoeae. Appl. Microbiol. 25: 550 to 552).

With the CTA system using CTA sugars, the incubation carried out in the air at 35 0C in order to promote the growth of the organism. While During growth, the organism produces an enzyme that breaks down the sugar. In the When the sugar is broken down, acidic end products are formed. The presence of an acid is determined by an indicator.

In the Kellogg system, however, the organism only grows incidentally, and the purpose is to remove the preformed enzyme that is already in place should discover.

The Kellogg system uses a small amount of substrate which are made from the same carbohydrate sugars used in the CTA tests. According to Kellogg, dense cell suspensions and a buffered saline solution are needed to discover the acidification faster. Confirmation that it is the culture N. gonorrhoeae is obtained within 5 hours of having a Purification plate successfully inoculated with a suspect colony of MTM and Has incubated for 18 to 24 hours.

Complications with the growth and non-growth carbohydrate digestion tests can be seen in the fact that maltose is common excess amounts of Contains contaminating, easily fermentable substances that are inapplicable Wise give positive reactions. It is also a subculture from isolated colonies from the MTM to the chocolate plates required for a heavy inoculum necessary to obtain sufficient growth.

When working according to Kellogg, only glucose, maltose, Sucrose and lactose. The fructose is omitted.

The sole degradation of glucose would identify an isolate as N.gonorrhoeae, whereas the additional breakdown of maltose, but no other carbohydrates, N. meningitidis would indicate.

Lactose is only broken down by N. lactamica, which makes it different from N. meningitidis is different. An improvement on Kellogg's method is of W. Jerry Brown (J.W. Brown, 1974, Modification of the Rapid Fermentation Test for Neisseria gonorrhoeae. Appl. Microbiol. 27: 1027-1030). Be after Brown the buffer salt solutions are modified and a heavier inoculum is used, which results in a method that is positive within 4 hours Identification leads. The Brownian modification of the Kellogg method is used the same naturally occurring substrates as Kellogg.

Recently, a method of positive identification within from 1 to 4 hours described (S.A. Morse and L.Bartenstein, 1976. Adaptation of the Minitek System for Rapid Identification of Neisseria gonorrhoeae. J. Clin. Microbiol.

3: 8-13). The Minitek company produces and sells microtiter plates, which contain shallow depressions. A disc is inserted into each of the wells, which contain natural substrates in a dry state. When it comes to carbohydrates in turn, they are glucose, maltose, lactose and sucrose, just as they are traditionally be used. The depressions be with a dense suspension inoculated by bacteria obtained from a cleaning plate.

The Minitek system relies on the production of acid from the carbohydrates and uses the sodium bicarbonate buffer to read negative reactions to facilitate. The in the publication by Morse et al., Loc. cit. used Slices use dextrose nitrate, dextrose, maltose, lactose and as substrates o-nitrophenyl-ß-D-galactopyranoside (ONGP).

With very high cell densities (greater than 5.0 x 109 colonies forming Units per milliliter) positive reactions can be achieved within 30 minutes read off. 90% of the isolates produce detectable acid within 4 hours from glucose. All isolates are identified within 6 hours. All Isolates identified with the CTA medium are also identified with the Minitek system. In addition, the Minitek procedure produces more isolates of N. gonorrhoeae and N. meningitidis as identified with the CTA medium.

Sometimes some false negative reactions are observed is often based on an inoculum with a low cell density. False negatives Reactions occur when inocula are taken directly from the Transgrow medium or the T-M plate, used in the initial isolation of the organism will. Morse solves this problem by returning isolates to a Spread on half of a GC agar plate and incubate overnight. The proof of work Minitek requires an inoculum from a cleaning plate that is 18 to 24 hours long incubated.

The ONPG reaction is usually called the equivalent of lactose fermentation considered. When an organism has the ability to hydrolyze the ONPG substrate and results in a color change, it is usually in CTA sugar fermentations for lactose lactose positive. N. lactamica is the only Neisseria species of Interest that can be distinguished from N. meningitidis and N. gonorrhoeae and the ability for hydrolysis of the synthetic ONPG and exhibits fermented lactose in a standard sugar fermentation test.

Usually three organisms can be grown on MTM medium, namely N.gonorrhoeae, N. meningitidis and N. lactamica.

Gonorrhoeae and meningitidis are commonly referred to as the two classified single pathogens of Neisseria.

The lactamic nerve is commonly isolated and is usually a Saprophyte. The distinction between the meningitid nerve and the lactamic nerve is based on the lactose breakdown or the ONPG test.

Both the meningitidis nerve and the lactamic nerve can contain glucose and maltose dismantle. Both are oxidase-positive organisms and gram-negative Diplococci that can grow on MTM medium. The discrimination test between The meningitidis and lactamica nerves are the ONPG test or the lactose breakdown test. Lactamic nerve can hydrolyze ONPG and break down or decompose lactose. N. meningitidis is for this not able.

Possibly other species of Neisseria can be found on MTM medium grow, but this is usually not the case.

However, if they grow on MTM, they will settle with the available standing technology readily from N.gonorrhoeae, N.meningitidis and N.lactamica differentiate by carbohydrate breakdown tests. Also Branhamella and Moraxella Species can grow on MTM medium, but this is usually not the case. If However, they can be transferred from N.gonorrhoeae, N.meningi! -tidis and N.lactamica through the The fact that these organisms do not break down carbohydrates and acid to produce. They were investigated to their enzymatic behavior with the invention to determine synthetic substrates. Morphologically, they are similar to Neisseria and oxidase positive.

The object on which the invention is based is achieved by a Device for the rapid identification of microorganisms, with a carrier base , several small or miniaturized reaction vessels on the support base are attached, a dried substrate in each reaction vessel, wherein the substrates are present in amounts of 25 to 50 nanomoles per reaction chamber and an amino acid coupled to a chromogen and a dried buffer in each of the reaction vessels, this being 1 to 500 nanomoles a buffer selected from Tris-HCl, Tris-Malate and phosphate buffer with K2HP04, KH2P04, as well as a polyvinyl alcohol, with the addition of disazo dye in aqueous solution with a polar solvent as a detection reagent and a inoculum to be identified and subsequent incubation at 350 during to the microorganism is identified within 5 hours, e.g. for 1 to 4 hours at 35 to 370C can be done by comparing the positive and negative reactions that occur with a given identification plate.

The object on which the invention is based is achieved in particular by a device for the rapid identification of Neisseria gonorrhoeae and Neisseria meningitidis, consisting of a support base, several small reaction vessels, attached to the support base, a dried substrate in each the reaction vessel in an amount of 25 to 50 nanomoles of an amino acid naphthylamide and approximately 1 to 500 nanomoles of buffer per reaction vessel.

The method according to the invention differs from the prior art in particular in that instead of naturally occurring substrates, synthetic Substrates are used or specific colorimetric substrates are used. The substrates are selectively hydrolyzed by enzymes during the incubation.

These enzyme activities have so far been used in microbiology not used in the identification of Neisseria. With the enzyme classes it can be aminopeptidase &, lipases, phosphatases, phosphodiesterases, Aryl amino acid hydrolases, aryl and alkyl amidases, and glycosidases.

With the help of the method according to the invention, one can faster gonorrhea than using the classic technique or the known fast working methods. In the method according to the invention, a sample is on a selective medium, such as for example the MTM medium. The purification stage of growth on a non-selective medium such as chocolate agar is not required. If you are on the selective medium growth discovered is inoculated on a synthetic substrate.

The present invention is based on enzymatic reactions that use chromogenic substrates. Those used in the process according to the invention synthetic substrates are available commercially. In the course of hydrolysis of the synthetic Substrates by the organism, a product is released by an indicator can be proven.

In the substrates used to practice the present invention it concerns the following: substrates detected enzyme-enzyme abbreviations 1) ß-Naphthyl-ß-D- ß-galactosidase BGAL gl actopyranosid 2) N-L-2-glutamyl-ß- # -Glutamylaminopepti- AGAM naphthylamide dase 3) L-hydroxyproline-ß-hydroxyproline-amino-OHPAP naphthylamide peptidase 4) L-serine-ß-naphthyl-serine aminopeptidase SAP amide Substrates Detected enzyme-enzyme Abbreviations 5) L-arginine-ß-arginine-aminopepti- AAP naphthylamide this 6) glycine-glycine-ß-glycyl-glycine-amino-GAP naphthylamide peptidase 7) ß-naphthyl-phosphate Acid phosphatase AP 8) ß-naphthyl valerate Valerase VAL 9) 4-methoxyleucine-ß- 4-methoxyleucine- 4-LEU naphthylamide aminopeptidase 10) Glycine-ß-naphthyl-glycine aminopeptidase GLY amid substrate stock solutions are prepared by placing the substrate in a 0.1 M solution of a salt of tris (hydroxymethyl) aminomethane (hereinafter abbreviated as "TRIS"), which contains 0.5% by weight of polyvinyl alcohol (PVA), and as in table B buffers shown to a given pH.

TABLE B Substrate TRIS salt preferred pH range pH 1 TRIS-HCl 6.8 5.0-7.9 2 TRIS malate 7.6 6.3-8.3 3 TRIS malate 7.2 6.3-8.0 4 TRIS malate 7.2 6.3-7.5 5 TRIS-Malate 8.0 6.8-8.4 6 TRIS-Malate 8.0 6.8-8.4 7 TRIS-HCl 5.5 5.2-5.8 8 TRIS malate 7.6 6.5-8.1 9 TRIS phosphate 7.6 6.0-8.1 10 TRIS phosphate 8.0 6.8 - 8.4 All substrate stock solutions have a concentration of 1 x 10 3 molar (M). 50 microliters each of the individual substrate solutions are in a small container. given to a test strip. Preferably is information printed on the test strip identifying the enzyme, that is to be formed in the adjacent small container. The final The concentrations of the reagents in each small container are 0.05 x 10 6 moles of the substrate and 5.0 x 10-b moles of Tris buffer. The strips are under vacuum or dried in the air below 50 ° C. After drying, the substrate will adhere and the disks or platelets on a porous backing layer.

Instead of the Tris buffer and the phosphate buffer, you can also use the Use the following buffer: 1. TES: N-tris (hydroxymethyl) methyl-3-aminopropanesulfonate 2. HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate 3. HEPPS: N-2-hydroxyethylpiperazine-N'-3-propanesulfonate 4. CHES: cyclohexylaminoethanesulfonic acid 5. CARBONATE: sodium or potassium carbonate 6. BORATE: sodium or potassium borate The buffers are prepared in aqueous solution, which contains 0.2% PVA, with a final concentration after adjusting the pH of 0.5 M.

Preferably, each of the ten substrates is divided into a separate one small container on a test strip as shown in the drawings is.

FIG. 1 shows a plan view of a biochemical test strip with ten individual reaction vessels (eight reaction vessels shown).

Figure 2 shows a longitudinal view of a test strip.

FIG. 3 shows a cross section through a test strip at which a single reaction vessel can be seen.

The test strip 10 consists of a rigid upper part 12 with several Openings 14 into which a reaction container or small container 16 is inserted, which is held in position by a rear part 18. The top 12 and the back 18 can be bonded to each other by glue. If both elements are suitable Material are made, for example from a thermoplastic resin, so can they can also be thermally welded to one another. The construction of the reaction vessel or small container can be seen in FIG. There it can be seen that the container 16 has a base 20 and a wall 22. In a preferred embodiment the wall is rounded. A flange 24 is molded around the base, which is preferably is provided with an upwardly bent peripheral ring. The flange engages between the upper part 12 and the rear part 18 and serves to hold the container in the to hold the assembled test strip in place. In the bottom part of the container 16, a disk or cushion 26 is attached. The disc is preferably made made of white cellulose wadding, but it can also be made of a polyvinyl chloride polymer or other suitable materials that are opposite to the material to be carried out Reaction are inert. In a particularly advantageous embodiment, the test strip is about 10 cm long and 2 cm wide. Each of the containers has a diameter of 5 mm and has a free height above the top of the disc of 5 mm.

The substrates are used at approximately 1.0 x 10 3M.

The exact concentration of each substrate should be such that at least 25 to 50 nanomoles are available per container.

The detection reagents are taken from the following immediately prior to each test described detection stock solution prepared.

The detection reagent is formed by adding 10.0 ml of the detection stock solution mixed with 0.4 g Fast Blue BB (sodium salt) Mixing do not expose to bright light; it has to follow within an hour used in mixing.

Detection stock solution: Tris base 175 g sodium lauryl sulfate 50 g hydrochloric acid 37% strength 55 ml of 2-methoxyethanol 500 ml of water 500 ml of 2-methoxyethanol added after Dissolution of the solids.

The following diazonium dye reagents were found to be specifically with the B-naphthyl released from the substrates by an enzyme or B-naphthylamine reacts: 1) Fast Red PDC 7) Fast Blue B 2) Fast Red B 8) Fast Scarlet CG 3) Fast Red AL 9) Fast Violet B 4) Fast Red TR 10) Kiazo Red RC 5) Fast Blue RR 11) Fast Black K 6) Fast Garnet ~~~~ 12) O-Diansidin The reaction of the above dyes with naphthylamine and naphthyl was in the laboratories of Applicant demonstrated and described in the literature (Biological Stains, H.J. Conn. Williamsl and Wilkins 1961). Dissolve each dye in the detection reagent stock solution with a content of 0.4 g / 10 ml. A typical example of the Use of one of the above dyes consists in using test strips as described produces, inoculates and incubates. Dissolve a 0.5 gram portion of any of the dyes described above or a 0.5 g portion of Fast Blue BB in 10 ml of detection reagent stock solution on.

Two drops of the above solution are placed in each of the small containers. The reaction is allowed to proceed for 10 minutes at room temperature. A change the color compared to the negative control is considered a positive reaction. The absence | a color change represents a negative reaction. The observed Sample is compared to Table A and the organism is determined by comparison.

In Table A below shows the first row to the right of each Organism the number of positive reactions and the number of tests in fraction form written, the second line shows the percentage of positive reactions and the third line indicates the reaction with a symbol. The symbol "-" stands for a predominantly negative reaction, the symbol + stands for a predominantly positive response and the symbol "v" means a variable response.

The identification of the microorganisms is carried out in | which one detects the chromogen released from the substrate by enzymatic cleavage. The detection range is 5 to 40 nanomoles of chromogen. The reaction typically occurs as follows: ß-naphthylamine + A-glutamic acid Fast Blue BB (colorless or pale yellow b) Diazo dye (dark purple or orange) The enzyme classes used in the test system are: 1. Glycosidases 2. Aminopeptidases (arylamidase) (hydrolytic aminotransferases) 3. Phosphatases 4. Carboxylases As far as the arrangement of the enzymes in relation to the bacterial cell is concerned, the following applies: the detectable enzymes can be either extracellular, periplasmic or cell-bound enzymes. Enzymes released by autolysis can also be detected.

The examinations used in the test system measure the sum of the Activities of a class of enzymes rather than a single unit of enzyme. A A change in color from pale yellow to purple or orange indicates a positive reaction.

The following examples assume that a single colony is 2 mm or more in size.

Example 1 This example illustrates one method of identification from N. gonorrhoeae.

Suspend at least 2 mm of growth from a single large one Colony of suspected N.gonorrhoeae in 0.5 ml sterile 0.85% sodium chloride with 0.05% calcium chloride and 0.05% sodium bicarbonate in water. The growth can from modified Thayer-Martin-Medium (MTM), Chocolate-Agar (CA), NYC-Mediut (NYC) or Transgrow Medium (TM) after 24 hours of incubation (primary isolation plate) can be obtained. One drop of suspension is used for a standard oxidase test and perform a standard gram stain. The rest is in the small containers inoculated onto the test strip to form one drop of suspension per container. Incubate place the test strip in a moist, covered plastic chamber for 1 hour 35 to 37 0C.

One drop is placed in each small container on the test strip Detection reagent. The reaction is allowed to proceed for 10 minutes at room temperature. The microorganism is identified by comparing the color of the reaction mass with table le A.

Example 1 2 This example explains an alternative way of working to identify N.gonorrhoeae.

One removes bacterial growth from the surface of an enriched one Chocolate agar plate, a Thayer-Martin medium (MTM) or a similar, for Neisseria allowed medium after soaking in 5.0% carbon dioxide for 18 to 24 hours clutivated for a long time at 35 ° C. It is suspended in 0.85% sodium chloride solution. The suspension is made with a 0.85% sodium chloride solution on a MacFarland No. 3 nephthalometer standard set. Then you give 20 to 50 microliters of this Suspension as an inoculum in each of the containers on the test strip for enzymatic use Analysis.

The test strip is incubated in a moist, closed chamber 1 hour at 35 to 37 ° C. A drop of the detection reagent is placed in each Given the container of the test strip. The reaction is left at room temperature for 10 minutes take place. The identification of the microorganisms is carried out by Compare the color of the reaction mass with Table A.

Example 3 This example illustrates an alternative procedure to identify a suspected N.gonorrhoeae colony.

In this procedure, the 0.1 M TRIS malate buffer is pH 8.0 replaced by a phosphate buffer with pH 7.3. The phosphate buffer contains per liter: K2HP04 ......................... 4.0 g KH2P04 ................... ...... 1.0 g polyvinyl alcohol (PVA) ........ 5.0 g Alternatively, the substrates can be dissolved in distilled water, wherein the buffer is introduced into the medium.

Remove a single colony of suspected N. gonorrhoeae a primary insulation panel, preferably an MTM panel. The colony diameter should be larger than 1.5 mm.

The colony is grown with vigorous agitation in a growth medium suspended, which contains the following components in one liter: proteose peptone No. 3 ...... 7.0 g glucose ......................... 4.0 g K2HP04 4.0 9 KH2P04. ........................ 1.0 g NaHC03 ................ 0.5 g IsoVital X (Difco, BBL) ......... 10.0 ml Das The above medium can be prepared by the method described by R.T. Jones and R.S. Talley (J. Clin. Microbiol. 1977, 5: 9-14) must be replaced. Any medium that has a fast Growth of Neisseria supported and colorless can be used will.

20 to 40 microliters of the inoculated medium are brought into contact with the substrate solutions.

TABLE A Organizational number B-GAL #GAM OHPAP AAP SAP GAP AP VAL 4-LEU GLY must under- seeks 1 2 3 4 5 6 7 8 9 10 N.gonorrhoeae 37 0/37 0/37 37/37 36/37 1/37 3/37 0/37 37/37 19/37 7/37 0 0 100 97.2 2.7 8.1 0 97.2 45.9 18.9 - - + + - - - + vv Meningitidis nerve 16 0/16 16/16 1/16 16/16 3/16 11/16 4/16 6/16 15/16 15/16 0 100 6.2 100 18.7 68.7 25 40 100 93.3 - + - + - vvv + + Lactamica nerve 15 15/15 0/15 15/15 14/15 14/15 14/15 14/15 5/15 15/15 14/15 100 0 100 93 93 93 93 33.3 100 93 + - + + + + v + + + M. mucosa 11 0/11 0/11 11/11 11/11 10/11 9/11 11/11 0/11 10/11 11/11 0 0 100 100 91 82 100 0 91 100 - - + + + + + - + + N.sicca 7 0/7 0/7 6/7 7/7 6/7 1/7 6/7 1/7 7/7 7/7 0 0 86 100 86 14 86 14 100 100 - - + + + v + - + + N.flavescens 5 0/5 0/5 5/5 5/5 5/5 5/5 0/5 0/5 5/5 5/5 0 0 100 100 100 100 0 0 110 100 - - + + + + - - + + TABLE A Organizational number B-GAL #GAM OHPAP AAP SAP GAP AP VAL 4-LEU GLY must under- seeks 1 2 3 4 5 6 7 8 9 10 N.flava 17 0/17 0/17 17/17 16/17 15/17 4/17 2/17 17/17 16/17 17/17 N.perfalva 0 0 100 94 88 23 12 100 94 100 N. subvlava - - + + + vv + + + b.catarrhalis 8 0/8 0/8 3/8 8/8 8/8 8/8 0/8 7/8 8/8 8/8 0 0 37.5 100 100 100 0 87.5 100 100 - - v + + + - + + + TM-1 6 0/6 0/6 6/6 6/6 0/6 6/6 0/6 0/6 6/6 6/6 0 0 100 100 0 100 0 0 100 100 - - + + - + - - + + TABLE A Organizational number B-GAL #GAM OHPAP AAP SAP GAP AP VAL 4-LEU GLY API mus under- CODE seeks 1 2 3 4 5 6 7 8 9 10 M-3 3 0/3 0/3 2/3 3/3 1/3 1/3 3/3 2/3 3/3 3/3 No. positive 0 0 67 100 33 33 100 67 100 100% positive - - v + vv + v + + reaction N-4 6 0/6 6/6 0/6 6/6 0/6 0/6 0/6 0/6 0/6 0/6 " 0 100 0 100 0 0 0 0 0 0 " - + - + - - - - - - " N-5 6 0/6 0/6 6/6 6/6 6/6 6/6 0/6 0/6 6/6 6/6 " 0 0 100 100 100 100 0 0 100 100 " - - + + + + - - + + " N-6 3 0/3 0/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 " 0 0 100 0 0 0 0 0 100 0 " M.osloen- 6 0/6 0/6 1/6 6/6 0/6 6/6 6/6 6/6 6/6 6/6 " sis 0 0 16.6 100 0 100 100 100 100 100 " - - v + - + + + + + " M.nonlique- 5 0/5 0/5 0/5 5/5 3/5 5/5 5/5 2/5 5/5 5/5 " faciens 0 0 0 100 60 100 100 40 100 100 " - - - + v + + + + + + " TABLE A Organizational number B-GAL #GAM OHPAP AAP SAP GAP AP VAL 4-LEU GLY API mus under- CODE seeks 1 2 3 4 5 6 7 8 9 10 M. lacuna- 4 0/4 0/4 0/4 0/4 0/4 0/4 4/4 0/4 4/4 4/4 No. positive ta 0 0 0 0 0 0 100 0 100 100% positive - - - - - - + - + + reaction M.phenyl- 2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 2/2 2/2 0/2 " pyruvica 0 0 0 0 0 0 0 100 100 0 " - - - - - - - + + - " M.kingii 2 0/2 0/2 2/2 0/2 0/2 2/2 2/2 2/2 2/2 2/2 " 0 0 100 0 0 100 100 100 100 100 " - - + - - + + + + + " L eerseite

Claims (1)

Claims 1. Method for the rapid identification of microorganisms, characterized in that a predetermined amount of a saline solution, the one microorganism to be identified is added to a solution containing a diazonium dye detection reagent in aqueous solution with a buffer and a substrate at about 35 0C Incubated for up to 5 hours and the positive and negative reactions with a Identification panel compares, where the substrate is selected from: 1) ß-naphthyl-ß-D-galactopyranoside 2) N-L-7-glutamyl-ß-naphthylamide 3) L-hydroxyproline-R-naphthylamide 4) L-serine-ß-naphthylamide 5) L-arginine-ß-naphthylamide 6) glycine-glycine-R-naphthylamide 7) ß-naphthyl-phosphate 8) β-naphthyl valerate 9) 4-methoxyleucine-R-naphthylamide 10) giycin-13-naphthylamide 2. The method according to claim 1, characterized in that a solution is used which is a detection reagent made from Fast Blue BB (sodium salt) and a stock solution of Tris base, sodium lauryl sulfate, 37% hydrochloric acid, 2-methoxyethanol and water, a buffer selected from Tris-HCl, Tris-malate, monopotassium phosphate and dipotassium phosphate in a polyvinyl alcohol and the substrate. 3. Device suitable for performing the method according to claims 1 or 2 for quick identification of Neisseria gonorrhoeae in a sample, which is only one of the microorganisms N. gonorrhoeae, N. meningitidis or N. lactamica includes, characterized by: a) a support base; b) at least two reaction vessels, which are fixed on the support base; c) a dried substrate that is in each the reaction vessel is arranged and which is once I3-naphthyl-ß-.D-galactopyranoside and also other is L-hydroxyproline-ß-naphthylamide; and d) a dried one Buffer, which is arranged in each reaction vessel, whereby by using the Device a negative reaction in the reaction vessel containing the ß-naphthyl-ß-D-galactopyranoside contains and a positive reaction in the reaction bed containing the L-hydroxyproline-ß-naphthylamide contains, N. gonorrhoeae appears. 4. Apparatus according to claim 3, characterized in that it is in the case of the dried buffer, which is arranged in each reaction vessel, to make Tris-HCl, Tris-malate, monopotassium phosphate and dipotassium phosphate in a polyvinyl alcohol. 5. Apparatus according to claims 3 or 4, characterized in that it has at least three reaction vessels and that in each reaction vessel as a dried substrate at least one of the following compounds: 1) ß-naphthyl-ß-D-galactopyranoside; 2) N-L - \ - glutamyl-β-naphthylamide; 3) L-hydroxyproline-β-naphthylamide; is contained, whereby by using the device a positive reaction in the reaction container, which contains the L-hydroxyproline-ßnaphthylamide and a negative reaction in the two other reaction vessels Neisseria gonorrhoeae is shown. 6. Apparatus according to claim 5, characterized in that the first Reaction vessel ß-Naphthyl -ß-D-galactopyranoside, the second reaction vessel N-L - \ - glutamyl-ß-napthylamide and the third reaction vessel L-hydroxyproline-ß-naphthyl contains amide, whereby by using the device a positive reaction in the third Reaction vessel and a negative reaction in the first and second reaction vessels Neisseria gonorrhoeae is shown. 7. Apparatus according to claim 6, characterized in that in each Reaction container a buffer is arranged. 8. Apparatus according to claim 7, characterized in that it is in the buffer to Tris-HCl, Tris-Malate, monopotassium phosphate and dipotassium phosphate in a polyvinyl alcohol. 9. Device suitable for performing the method according to claims 1 or 2, to distinguish between Neisseria gonorrhoeae, N. meningitidis and N. lactamica in a sample that contains only one of these microorganisms includes, characterized by: a) a support base; b) a first reaction vessel, which is attached to the support base and contains β-naphthyl-β-D-galactopyranoside; c) a second reaction vessel attached to the support base and N-L-X-glutamyl-ß-naphthylamide contains; d) a third reaction vessel which is attached to the support base and contains L-hydroxyproline-ß-naphthylamide; and e) a dried buffer which is arranged in each reaction vessel, and which is Tris-HC1, Tris-Malate, Monopotassium phosphate and dipotassium phosphate in a polyvinyl alcohol; whereby a negative reaction in the first and second reaction vessels by using the device and a positive reaction in the third reaction container N. gonorrhoeie is displayed, N. meningitidis is indicated by a positive reaction in the second reaction container and by a positive reaction alone in the first reaction container N. lactamica are displayed. 10. Device suitable for performing the method of claims 1 or 2 for quick identification of Neisseria gonorrhoeae and Neisseria meningitidis, characterized by: a) a support base; b) several reaction vessels, which are fixed on the support base; c) 1 to 500 nanomoles of buffer per reaction vessel, selected from Tris-HCl, Tris-Malate and Phosphate Composition with a content on K2HP04, KH2P04 and a polyvinyl alcohol; and d) a dried substrate, which is arranged in each reaction vessel and per reaction vessel from 25 to 50 nanomoles of one of the following compounds consists: 1) ß-Naphthyl-ß-D-galactopyranoside 2) N-L -) - Glutamyl-ß-naphthylamide 3) L-hydroxyproline-ß-naphthylamide 4) L-serine-ß-naphthylamide 5) L-arginine-ß-naphthylamide 6) Glycine-glycine-ß-naphthylamide 7) R-naphthyl-phosphate 8) ß-naphthyl valerate 9) 4-methoxyleucine-ß-naphthylamide 10) glycine-ß-naphthylamide . 11. The device according to claim 10, characterized in that it Has 10 reaction vessels.