FI57128C - SAETT ATT IDENTIFIED MICROORGANISM - Google Patents

Description

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Patent · och ragltteratyralaan '/ Ameicu uttagd oeh uti ^ kriftM puMiewvd 29.02.80 (32) (33) (31) Requested «tuolkuu» —Rcgird priorltet (71) Qrion-Yhtyma, Oy, Nilsiänkatu 10-lU, 00510 Helsinki 51, Suomi-Finland (Fl) (72) Jouko Savolainen, Järvenpää, Suomi-Finland (FI) (7 * 0 Fermion Oy, Patent Department (51+) The way to identify microorganisms - Sätt att identifiera mikroorganismer

The object of the invention is to present a simple way of performing the qualitative identification of microorganisms in a compartment dish by means of carbohydrate and nitrogen metabolism tests, a urea test and morphology. In this context, microorganisms, i.e. microbes, mainly mean fungi, yeasts and bacteria.

In medicine, veterinary medicine, the food and fermentation industries, the identification of microorganisms is important. The identification of microbes is essential when choosing a method for destroying microorganisms and regulating their growth conditions. Identification of disease-causing microorganisms is important to determine their pathogenicity and to select the appropriate method of treatment. In the food and fermentation industry, microorganisms are beneficial organisms, but they often also occur as contaminants. These pollutants need to be identified in order to find out their,:, W, ...

, V · 1 2 57128 primary sources and in order to select the right methods to prevent their growth and spread.

The identification of microbes is mainly based on the morphology of the microbial growth, the ability to use different carbohydrates and nitrogen sources, different growth conditions and the exploitation of certain other properties.

Carbohydrate assimilation tests as well as nitrogen metabolism tests can be performed in three different ways. The methods are the auxanographic method, the Wickerham broth tube method, and the oblique surface method developed from the Wickerham broth tube method.

These methods are described in Manual of Clinical Microbiology 2nd ed. 1974, pp. 491-507 and Journal of Clinical Microbiology 2, No. 1, 1975, pp. 21-34.

The above methods and several of their applications have certain disadvantages. The methods require a lot of material in the form of test tubes, plates, substrates, etc., are time and space consuming as well as laborious to perform. The disadvantage is also the growing risk of contamination with a large number of materials and work steps.

In order to eliminate these disadvantages, a method according to the invention has been developed, in which all the necessary tests, with the exception of fermentation tests, are performed on the same tray, whereby material consumption, manufacturing time and work and work steps can be significantly reduced.

The essential object of the present invention is to provide a simple way to perform the identification of a microorganism in a tray dish on the basis of carbohydrate and nitrogen metabolism tests, urea test and morphology. All experiments are performed on the same tray, one of which is reserved for the morphological test. This includes the only complete solid medium in the method and the other wells in the tray plate contain carbohydrate and nitrogen metabolism assays, as well as various solid agar media for the urea assay, which may also include an indicator and buffer.

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The method according to the invention is characterized in that the solid agar media used in the carbohydrate and nitrogen metabolism experiments and in the urea experiment do not contain the nutrients necessary for the growth of the microorganism before the start of the experiment. These are added to the compartments only after inoculation of the test microorganism in the form of tablets. The tablets contain pharmacologically acceptable, conventional excipients and binders as well as carbohydrates and nitrogen sources known per se for the identification of the microorganism. In addition, the tablets contain various nutrients necessary for the microorganism, suitably supplementing the carbohydrates and nitrogen "" sources in the tablet. For example, in the case of a carbon source, the supplemental nutrient is containing nitrogen sources, and in the case of a nitrogen source, the supplementary nutrient is containing carbon sources.

The device used in the invention is a sterile rectangular lid with a lid, which is divided into a plurality of rectangular compartments by means of rising, perpendicularly intersecting, impermeable partitions. Tray sizes and numbers may vary.

At the bottom of the compartments is a solid, slightly buffered or unbuffered agar medium suitable for each test to be performed, which may or may not contain an indicator, in which case the indicator may optionally be added after growth. The agar medium is inoculated with an appropriate amount of suspension of the microorganism to be tested. After inoculation, tablets containing the nutrients necessary for microbial growth, the test substances and pharmacologically acceptable excipients and binders are placed on the agar medium in a dish suitable for the plate known per se.

In order to be able to identify the micro-organism under examination in each case, the number and quality of the tests can be freely varied. This is achieved by varying the substrates used in the method of the invention as well as the tablets to be placed on these solid substrates.

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For example, the medium in the compartment reserved for the morphological study of yeasts is the well-known corn agar, but other low-nutrient media can also be used. These media for morphological studies are the only complete media used in the identification method, and tablets otherwise used in the invention are not added to these media. The tray fits just well in a coverslip, so microscopy, or morphological examination, can be performed directly on the plate and the preparation of separate microscope slides is avoided. To facilitate microscopy, known dyes can be added to the substrate.

For carbohydrate experiments and control experiment, the plates contain agar and indicator as well as buffer. As indicators, any pH indicator can be used which shows acid formation and a drop in pH below pH 5.5, for example bromocresol purple or bromothymol blue. The pH of the medium is adjusted halfway through the color change range of the indicator and buffered slightly against random pH fluctuations, e.g. with 5-10 mM phosphate buffer.

To test the uptake of nitrate and ammonium for nitrogen metabolism, the medium contains agar and an indicator such as bromothymol blue, the pH of which is adjusted between blue and yellow to, for example, pH 6.4.

For the urease assay, the medium contains agar and indicator. For example, phenol red can be used as an indicator. The pH of the medium is adjusted to pH 6.9.

The identification method requires only 4 different substrates, of which only one, i.e. the morphology substrate, is complete. Other media do not contain nutrients, so their composition effectively limits the growth of contaminants.

Since only 4 different substrates are required in the identification method, the workload of 5 57128 is considerably reduced compared to the known methods, in which a separate substrate has to be prepared for each experiment. At the same time, the durability of the substrates used in the method is improved and the number of work steps is reduced. The indicator does not necessarily need to be added to the medium, but can be pipetted onto the medium in the tray after growth.

The other necessary ingredients of the plate test trays, such as carbon source, nitrogen source, trace elements, and vitamins, are contained in the tablet, which together with the tray tray forms a growth-sustaining entity. Each tablet contains the nutrients necessary for the growth of the microorganisms, for example the ingredients of the Wickerham broth tube substrates, the test substances and the inert excipients of the tablet. Any tablet excipient used in pharmacology can be used as an excipient, which transfers the growth-critical ingredients to the medium during growth. Such fillers include, for example, cellulose and its derivatives. Binders can be used in the tablets to improve tablet cohesiveness and to facilitate preparation. For example, some plastic products used in pharmacology can be used as binders.

The percentage composition of the carbohydrate tablet is as follows: carbohydrate 12.7-25.4%, nutrients necessary for the growth of the microorganism, for example Difcon Yeast Nitrogen Base 4.3%, excipient 60.6-74.5% and binder 9.5% .

Various crystalline solid monosaccharides, disaccharides, oligosaccharides and polysaccharides can be used in carbohydrate tablets. Other solid carbon sources can also be used to make tablets.

For the control test, a tablet is prepared with all the ingredients except the carbohydrate. This tablet can also be used if the test is a non-crystallizing carbon source, for example ethanol or glycerol. In this case, a tablet without carbohydrate is placed in the test tray and a suitable amount of carbon source at a physiologically tolerable concentration is pipetted onto it to identify the microorganism 6 57128. For example, ethanol or glycerol can be used as a 20% solution, with a suitable amount for the experiment being 50 / tablet.

The composition of the nitrate, ammonium and urea tablet is respectively: nitrogen source 2.5%, nutrients necessary for the growth of the microorganism, for example Difcon Yeast Carbon Base 3.0%, excipient 85.0% and binder 9.5%.

The range of basic tablets per plate is as follows: nitrogen source positive control (NH 4, 1 SO 2, nitrate, carbohydrate control (without carbohydrate), glucose, maltose, sucrose, inositol, lactose, cellobiose, raffinose, melibiose, secoitol, erythritol, xylitol, xylitol Urea may be added to or removed from this list in such a way that the substances used in the test are best suited to identify the micro-organism under investigation.

It is preferable to place the tablets in place with a device designed and manufactured to fit exactly this cup. A tube is placed in the device for each tablet, so with one press, each tablet comes in the right place. Tablets are made to a certain weight by compressing the mass into a sufficiently hard tablet so that it does not disintegrate on the surface of the substrate after wetting.

The described method achieves considerable advantages. The use and handling of the material is reduced because all tests are on the same plate and not each test is in a separate test tube. Storage, working and rearing space is saved and testing is faster and more secure. Manufacturing is easier and faster because the bowl can be poured at once and in the same position. Only four substrates are needed instead of having to dispense a different medium into each test tube. Tablet making is simple and fast.

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There is no need for easily contaminated nutrient solutions, which can be difficult to prepare and sterilize, such as sterilizing sugar solutions by filtration. The tablets do not necessarily need to be sterilized as long as they are prepared using the necessary aseptic procedures. At the same time, drying the filter paper discs, pipetting solutions, etc. is avoided. The risk of contamination is reduced because the system, with the exception of the morphology medium, does not contain any complete medium. Plates and tablets can be stored at room temperature. In addition, the identification system is fast. The results can be read four days after inoculation of the dish.

The following application examples for yeasts, fungi and bacteria illustrate the use of the invention.

Example 1

From a clinical sample, yeast was cultured on Sabouraud agar to a pure culture. The suspension was prepared in sterilized distilled water from 1-2 yeast colonies. The suspension was inoculated with a needle into yeast corn agar by scraping the surface broken from side to side. One side of the scraper was covered with sterile coverslip and the other side was allowed to remain as is. Two drops of the same yeast suspension were dropped into each of the other compartments of the multi-compartment dish with a Pasteur pipette. The yeast suspension was stored at room temperature for future use. The tablets, which contain the nutrients necessary for the growth of the yeasts, the test substances, the fillers and the binders, were then placed in their own compartments on the surfaces of the substrates with a custom-made applicator 11a. The following compounds, glucose, maltose, sucrose, inositol, lactose, cellobiose, raffinose, melibiose, erythritol, xylose, dulsitol, trehalose, ammonium, nitrate and urea were tested for yeast identification. The dish was transferred to a 25 ° C oven for growth in the correct orientation.

After two and three days, the corn agar culture was microscopied directly from the plate at 700x magnification under bright field illumination. The result was that the test yeast formed a hyphae, · but chlamydospores and arthrospores were absent. The following results were obtained from the other tests: after 3 days of growth of β 57128, the nitrogen positive control was positive. The yeast grew on the substrate and the color of the substrate was yellow, as it must always be. The nitrate was negative, i.e. the color of the substrate was yellow. The carbohydrate control was dark green, i.e. the original color, as it should be. Of the sugars, glucose, maltose, sucrose, xylose, and trehalose were positive. Other sugars and urea were negative.

Based on these results, the yeast could have been Candida neoformans or Candida parapsilosis. The fermentation experiment confirmed that the yeast was Candida parapsilosis.

Example 2

Yeast was isolated from the milk of a cow with long-term mastitis and cultured clean. The method described above was used to identify yeast. The initial steps were the same as in the previous example.

In the printing step, the culture was microscopied directly from corn agar after 2 days of growth. Yeast formed a hyphae and a pseudo-hyphae, as well as arthrospores, which are formed by breaking the tips of the hyphae. Nitrogen and carbohydrate controls gave the correct reaction, indicating that the plate and sample were useful.

The result of the nitrate test was negative. Of the sugars, glucose, lactose, xylose, and dulsitol gave a positive result. Maltose, sucrose, inositol, cellobiose, raffinose, melibiose, erythritol, and trehalose were weakly positive. Urea was positive.

Based on these results, yeast was identified as Trichosporon cuta-neumi see i.

Example 3

Yeast was isolated from contaminated fresh orange juice and cultured. Yeast was identified by the system described. The initial steps were the same as in the previous examples.

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On corn agar, the yeast formed a pseudohyph. Other results after 4 days were as follows: controls were correct; nitrate was positive, of sugars glucose, maltose, sucrose, cello-biose, raffinose, erythritol and trehalose were clearly positive, xylose was weakly positive and inositol, lactose, melibiose and dulcitol were negative. Urea was positive.

Based on these results, the yeast was identified as Hansenula anomaly.

^ Example 4

The identification method described examined the ability of Escherichia coli (serogroup 0149) to assimilate carbohydrates and reduce nitrate, as well as urease production.

Escherichia coli was grown overnight in broth at 37 ° C. The culture was isolated from the medium by centrifugation and suspended in sterile distilled water to the original volume. As in the identification of yeasts, one drop of suspension was dropped on each medium of the dish, after which the corresponding tablets were placed in place. The plates were grown overnight at 37 ° C. The results were read in the same way as for the identification of yeasts.

Escherichia coli (serogroup 0149) reduced nitrate to nitrite (red color with the addition of a drop of sulphanilic acid and alpha-naphthylamine), - did not form urease, assimilated glucose, maltose, lactose, raffinose, melibiose, xylose and trehalose; did not assimilate sucrose, inositol, cellobiose, erythritol, or dulsitol. Positive and negative reactions were clear and corresponded to those reported in the literature.

The identification method is suitable for studying the corresponding characteristics required for the identification of species of Escherichia coli and in particular of the family Enterobacteriaceae.

Example 5

The yeast identification method described above was used to determine the usefulness of carbohydrates and nitrate in species of the genus Strepto-myces, as well as

Claims (1)

10 5 71 2 8 for the study of urease production for identification. Streptomycete cultures isolated from lake bottom mud and preserved with sodium caseinate agar surface were suspended in distilled water as in the case of yeasts. The suspension was added dropwise to each agar surface and the corresponding tablets were placed in place. The plate was kept at 25 ° C for 10 days, after which the results were read. The studied strain grew on corn agar and formed hyphens as well as small green colonies. The strain used nitrate efficiently but weakly hydrolyzed urea. In addition, the strain assimilated all 12 carbohydrates, i.e., grew and produced acid with all carbohydrates studied, which were glucose, maltose, sucrose, ino-Sitol, lactose, cellobiose, raffinose, melibiose, erythritol, xylose, dulcitol, and trehalose. The positive results for carbohydrates and nitrate were very clear. Simple method for the qualitative identification of micro-organisms as compartments 1 and on the basis of carbohydrate and nitrogen metabolism experiments, urea experiment and morphology, all experiments being performed simultaneously as compartments 1 and 11a, one of which is reserved for morphological experiment and contains the only complete solid medium of the method and in which the other compartments contain carbohydrate and nitrogen metabolism tests and various solid agar media for the urea test, which may also contain an indicator and a buffer, characterized in that the solid agar media used in the carbohydrate and nitrogen metabolism tests and the urea test do not contain essential nutrients, but these are added to the compartments after inoculation of the test micro-organism in the form of tablets containing any pharmacologically acceptable, conventional excipients and binders and Kullo carbohydrates and nitrogen sources known per se suitable for the identification of the microorganism and nutrients necessary for the growth of the microorganisms, in which case the latter are suitably supplemented.