WO1995003424A1

WO1995003424A1 - Culture medium for the simultaneous detection of coliform bacteria and escherichia coli

Info

Publication number: WO1995003424A1
Application number: PCT/EP1994/002240
Authority: WO
Inventors: Rolf Ossmer
Original assignee: Merck Patent Gmbh
Priority date: 1993-07-21
Filing date: 1994-07-08
Publication date: 1995-02-02
Also published as: EP0710291A1; DE4324392A1; JPH09501314A

Abstract

The invention relates to culture media for the simultaneous detection of coliform bacteria and E. coli and the use of these media to detect coliform bacteria and/or escherichia coli in examination samples. Besides the customary components, e.g. complex bases of nutritive media, salts to adjust the pH and ionic strength, the culture media of the invention also contain chromogenic substrates for the β-galactosidase and the β-glucuronidase which contain two differently substituted indolyl derivatives as chromogenic radicals. The media of the invention are solidified for use by, for example, the addition of gelling agents.

Description

Culture medium for the simultaneous detection of coliform bacteria and Escherichia coli

description

The invention relates to nutrient media for the simultaneous detection of coliform bacteria and E. coli, and the use of these media for the detection of coliform bacteria and / or Escherichia coli in test samples.

The detection and determination of the bacterial count of E. coli is an extremely important parameter for the assessment of the microbiological quality of food and water: the presence of E. coli is an indication of faecal contamination. The detection and determination of coliform bacteria as indicator germs is similarly important, since their presence indicates poor hygiene. This results in the need to be able to detect both the presence of E. coli and coliform bacteria in a sample and to be able to determine the respective number of bacteria.

In addition to the standard methods for the detection of coliform bacteria and E. coli, which are very laborious and time-consuming (up to 96 hours), the use of enzyme profiles for such detection methods was introduced.

It was found that the detection of β-glucuronidase activity using chromogenic or fluorogenic substrates is highly specific for E. coli. For the glucuronidase detection by FRAMPTON et al .; J. Food Prot. 51 402-404 (1988) and by WATKINS et al .; Appl. Environ. Microbiol. 54, 145-150 (1988) proposed the substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC). The blue reaction product (indigo) is easily recognizable on the culture medium regardless of the pH value and diffusion into the culture medium hardly takes place.

The presence of galactosidase is characteristic of the group of coliform bacteria. Suitable chromogenic or fluorogenic substrates are known; this includes 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-GAL), which was developed by MANAFI and KNEIFEL; Zentralbl. Hyg. 189, 225-234 (1989).

Further chromogenic and fluorogenic enzyme substrates suitable for the detection of β-glucuronidase activity and of galactosidase activity are:

4-nitrophenyl-ß-D-glucuronide and 4-methylumbelliferyl-ß-D-glucuronide (MUG) (detection of ß-glucuronidase activity) o-nitrophenyl-ß-D-galactopyranoside (ONPGAL), p-nitrophenyl-ß- D-galactopyranoside (PNPGAL) and 4-methylumbelliferyl-ß-D-galactopyranoside (MUGAL)

(Detection of galactosidase activity)

So that both the coliform bacteria and E. coli can be characterized from mixed cultures without additional effort, solid culture media should at the same time allow the detection of the glucuronidase activity in addition to the galactosidase activity. Only one of the substrates X-GAL and X-GLUC can be used because the products of both detection reactions are identical. For this reason, for example, glucuronidase detection with X-GLUC was combined with detection of lactose utilization by means of pH indicators (MATNER et al .; J. Food Prot. 53, 145-150 (1990) and DARTORY and HOWARD; Lett Appl. Microbiol. 15 (6). 273-276 (1992)). Other authors have published the

Galactosidase detection with X-Gal and glucuronidase detection combined with the fluorogenic substrate MUG (MANAFI and KNEIFEL; Zentralbl. Hyg. 189, 225-234 (1989).

In both cases, the detection of the second enzyme activity is due to the non-specificity of the pH change or the difficulties in detecting fluorescence from methylumbelliferone (working in the darkened room under UV light; necessity to neutralize the medium for the detection with alkali; quenching Reactions in the detection of fluorescence; strong diffusion of the fluorescent dye in the

Culture medium) has considerable disadvantages. So it turns out Task to provide improved nutrient media which allow the specific detection of both the glucuronidase activity and the galactosidase activity on the same nutrient medium.

The connection is under the registration number CAS 1338 182-21-5

6-chloro-3-indoiyl-β-D-galactopyranoside (Salmon-Gal / Rose-Gal) known. It has been found that this compound can be used as a chromogenic substrate for galactosidase, it also being possible to use 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC) as a chromogenic substrate for glucuronidase . The use of these two substrates allows a clear differentiation of the two enzyme activities on the nutrient medium: galactosidase produces a red color, glucuronidase a turquoise-blue color; Bacterial colonies in which both enzyme activities are present are colored dark purple. The colors are easily recognizable on the culture medium regardless of the pH, they do not diffuse into the culture medium, but remain in the colonies. The colors remain stable for several days.

The invention relates to nutrient media for the detection of Escherichia coli and / or coliform bacteria essentially consisting of complex nutrient media, salts for adjusting pH and ionic strength, chromogenic substrates for β-galactosidase and for β-glucuronidase and, if appropriate, other conventional constituents, characterized in that two differently substituted chromogenic residues for the β-galactosidase and β-glucuronidase substrates

Indolyl derivatives are included, it being possible to additionally add gelling agents to the nutrient medium.

The invention further relates to dry preparations for the preparation of nutrient media according to the invention.

The invention relates to the use of nutrient media with the composition given above for the detection of Escherichia coli and / or coliform bacteria in a test sample. The invention relates to methods for the detection of Escherichia coli and / or coliform bacteria, characterized by the following method steps: a) inoculating a nutrient medium with an examination sample, the nutrient medium having the composition according to the invention; b) incubate the inoculated nutrient medium until bacterial colonies become visible; c) Observation of the colored bacterial colonies.

The basic constituents contained in the nutrient media according to the invention are known to the person skilled in the art. These include gelling agents, for example agar-agar. For certain applications it is customary to prepare a nutrient medium without agar agar and then to use this to carry a carrier, e.g. Nutrient carton to soak to create a solid culture medium.

According to the invention, the term gelling agent is understood in a broader sense to mean additives with the aid of which a nutrient solution is brought into a solid or semi-solid form and which, if appropriate, can also be added subsequently. In addition to the other additives mentioned for this purpose, those skilled in the art are aware of them.

Furthermore, the nutrient media according to the invention contain complex nutrient soil components, such as yeast or meat extract, peptones from various protein-containing products. Further components of the nutrient media according to the invention are neutral salts for adjusting the ionic strength, e.g. NaCI, as well as buffer salts for adjusting the pH, e.g. primary and secondary potassium or sodium phosphate. A pH between 6 and 8 is preferably set.

The chromogenic substrates used according to the invention contain differently substituted indole derivatives as chromogenic residues, which are each bound in a glycosidic bond to galactopyranose or glucuronic acid. The indole derivatives are selected so that the resulting indigo derivatives have different colors. gene. The enzymatic cleavage thus produces two differently substituted indoxyl derivatives, each of which oxidatively dimerizes to differently colored indigo derivatives under the influence of atmospheric oxygen. A suitable combination of two such dyes is mentioned as an example. However, further indoxyl derivatives are known from the chemistry of the indigo precursors which react to give differently colored indigo dyes and which can be used in a similar manner for the purpose according to the invention. For example, indoxyl and 5,7-dibromoindoxyl give a blue, 6-bromoindoxyl a purple indigo derivative.

In order to stimulate the expression of the enzymes on which the detection reactions are based in the bacteria, enzyme inducers are added; for example, isopropyl-β-D-1-thiogalactopyranoside (IPTG) increases galactosidase activity and methyl-β-D-glucuronide increases glucuronidase activity.

Further additives known to the person skilled in the art in various nutrient media are growth factors and inhibitors.

Growth factors complement the nutrient media, which include, for example, organic compounds such as pyruvic acid

(Pyruvate) or L-tryptophan. The addition of L-tryptophan makes it possible to carry out the indole detection specific for E. coli in addition to the detection of glucuronidase and galactosidase activity. - Many test samples contain unwanted accompanying germs, the growth of which can be reduced by growth inhibitors. For this purpose, for example, detergents such as bile salts, lauryl sulfate or Tergitol® and the more potent novobiocin are known to the person skilled in the art. Nutrient media containing novobiocin suppress the accompanying flora very strongly; the inhibitory effect of the other inhibitors mentioned is less. Nutrient media containing novobiocin or similarly strong inhibitors are particularly suitable for investigations on samples that are heavily contaminated with accompanying germs. For this purpose, lauryl sulfate and novobiocin are preferably combined. In contrast, nutrient media are the only weaker-acting inhibitors included, particularly suitable for samples with subletally damaged germs. The use of lauryl sulfate is preferred for this purpose.

Commercial nutrients are often used as dry preparations

(Dry culture media) are provided, which are mixed with water, dissolved and sterilized. Such products and their use are familiar to the person skilled in the art. In a preferred embodiment, the solid constituents of the nutrient media according to the invention are mixed together to form a dry powder and, if appropriate, additionally granulated. These processing steps are familiar to the person skilled in the art.

Culture media, which are initially prepared without the addition of a gelling agent, can serve, after sterilization, for example to soak sterile cardboard disks, on which filter media are then placed for incubation. Such methods are also known from the prior art.

The presence of E. coli is considered an indication of faecal contamination of the sample; coliform bacteria are an indication of poor hygiene. Liquid or solid samples can be examined:

Liquid samples such as well, surface, drinking water or drinks are applied directly to the nutrient medium. If the sample contains a very high number of bacteria, it becomes sterile

Saline or pre-diluted with sterile peptone water. In general, 100 μl of liquid inoculum are applied and distributed on the culture medium.

If the sample contains very few germs, it can pass through

Centrifugation be enriched before application: A larger volume, for example a few milliliters, is centrifuged and the bacterial precipitate is taken up in a smaller volume of sterile peptone water and applied as an inoculum. Another common method for enriching bacteria from liquid samples, such as drinking water, is filtration. A larger volume of the sample is sucked through a membrane filter by means of negative pressure; membrane filters made of cellulose nitrate with a pore size of 0.45 μm are common. Then the filter is on the

Culture medium laid up and incubated.

Solid samples can be smeared directly on the nutrient medium or rinsed or homogenized with sterile saline or with sterile peptone water, the suspension then being applied to the nutrient medium.

For example, solid samples can be mixed with ten parts of buffered peptone water. Devices that are known under the name "stomacher" can be used for this. The suspension obtained in this way or a dilution thereof is then applied to the nutrient medium.

The details of such processes and possible process variants are familiar to the person skilled in the art and can be found in manuals such as

Compendium of Methods for the Microbiological Examination of Foods (M. L. Speck (ed.); (1984) American Public Health Association). After the inoculation, the nutrient media are incubated, the incubation temperature typically being between 25 ° C. and 50 ° C. The incubation period is typically between 12 and 48 hours. The person skilled in the art varies the incubation conditions, for example, depending on the sample examined. The following incubation conditions are preferred: The inoculated culture media are incubated under aerobic conditions at 35-37 ° C. for 24 hours. If the reaction is not very pronounced or to ensure a negative result, the incubation period can be extended by 24 hours. In general, the results can be evaluated after 24 hours of incubation.

According to the invention, nutrient media with the following constituents and concentration ranges are preferred: meat extract (2-5 g / l), NaCl (1-10 g / l), sodium pyruvate (0.5-2 g / l), potassium di-hydrogen phosphate (1-3 g / l), di-potassium hydrogen phosphate (2-4 g / l), Salmon-Gal (0.03-0.2 g / l), IPTG (0.03-0.2 g / l), X-GLUC (0.01-0.2 g / l), methyl-β-D-glucuronide (0.03-0.2 g / l), sodium lauryl sulfate (0.03-0.2 g / l), L-tryptophan (0.5-2 g / l) and optionally agar agar (8-20 g / l), and optionally Novobi biocin (sodium salt; 0.003-0.01 g / l). Particularly preferred nutrient medium compositions are described in Examples 1 and 2.

The composition of the nutrient media according to the invention allows all active constituents to be mixed together to form a powder mixture or granules. This mixture can be dissolved directly in water and autoclaved.

The use of culture media with a composition according to the invention normally allows the results to be read after only 24 hours, even if the bacteria are subletally damaged. In contrast, in known methods, e.g. incubated for 48 hours using EMX agar (Manafi, M. and W. Kneifel (1989) Zentralbl. Hyg. 189, 225-234).

Even without further explanations, it is assumed that a person skilled in the art can use the above description in the broadest scope. The preferred embodiments are therefore only to be understood as a descriptive disclosure, and in no way as a limitation in any way.

The complete disclosure of all applications, patents and publications listed above and below, and the corresponding application (s) DE 4324 392, filed on July 21, 1993, are incorporated by reference into this application.

Examples

The following examples are intended to explain the subject in more detail; they do not represent any restriction of the subject of the invention general working methods known to the person skilled in the art are not explained in more detail; Manuals such as Compendium of Methods for the Microbiological Examination of Foods (ML Speck (ed.); (1984) American Public Health Association) describe such methods.

Example 1: Nutrient medium with low inhibitory activity against gram-positive bacteria

Component quantity

(g i)

Meat extract 3.0

NaCI 5.0

Sodium pyruvate 1.0

Potassium di-hydrogen phosphate 1.7 di-potassium hydrogen phosphate 3.0

Salmon Gal 0.1

IPTG 0.1

X-GLUC 0.1

Methyl-β-D-glucuronide 0.1

Sodium lauryl sulfate 0.1

L-tryptophan 1.0

Agar agar 10.0

The ingredients are dissolved together in one liter of demineralized water and the nutrient medium is autoclaved (15 minutes at 121 ° C). The culture medium has a pH of 6.8 + 0.2; if necessary, the pH can be adjusted with sterile-filtered sodium hydroxide solution or hydrochloric acid. The nutrient medium is then cooled to 45 ° C in a water bath and e.g. poured into petri dishes. The plates of this culture medium are clear and colorless.

This medium enables a very high recovery rate even with sublethally damaged bacteria. Example 2: Nutrient medium with increased inhibitory action against gram-positive bacteria

Component quantity

(g / i)

Meat extract 3.0

NaCI 5.0

Sodium pyruvate 1.0

Potassium di-hydrogen phosphate 1.7 di-potassium hydrogen phosphate 3.0

Salmon Gal 0.1

IPTG 0.1

X-GLUC 0.1

Methyl-β-D-glucuronide 0.1

Sodium lauryl sulfate 0.1

L-tryptophan 1.0

Novobiocin (sodium salt) 0.005

Agar agar 10.0

The medium is prepared as described in Example 1.

The addition of Novobiocin makes it possible to detect E. coli or coliform bacteria even in the presence of a strong accompanying flora.

Example 3: Culture of various bacteria on a nutrient medium according to Example 1

Bacterial strains 1. Escherichia coli ATCC 25922

2. Citrobacterfreundii ATCC 6750

3. Enterobacter aerogenes ATCC 13048

4. Klebsiella pneumoniae ATCC 13883

5. Salmonella sp. Serotype enteritidis ATCC 13076 6. Shigella flexneri ATCC 12022

7. Bacillus cereus ATCC 11778

8. Bacillus subtiiis ATCC 6633

9. Enterococcus faecalis ATCC 19433

10. Enterococcus faecium ATCC 6569 1. Listeria monocytogenes ATCC 19118

12. Salmonella sp. Seratum anatum ATCC 9270

One inoculation loop of bacteria from a pre-culture of the respective strains 1-12 is suspended in 5 ml of sterile peptone water. 100 μl of each suspension are dripped onto a Petri dish containing a nutrient medium according to Example 1 and distributed. The inoculated culture media are incubated under aerobic conditions at 35 ° C for 24 hours. The growth, color of the colonies and the result of the detection reactions with Salmon-Gal and X-GLUC are then evaluated and the indole reaction is carried out. The following results were found:

Germ growth color of the detection reaction: (number) colonies Salmon-Gal X-Gluc indole

1. good dark blue-violet + + +

2. good red +

3. good red +

4. good red +

5. good colorless -

6. good colorless. . .

7. no

8. slightly colorless. . .

9. light pink +/- 10. light pink +/- 11. no 12. good light blue-turquoise +

Example 4: Culture of various bacteria on a nutrient medium according to Example 2

The experiment described in Example 3 was repeated with nutrient medium according to Example 2; the following results were found:

Germ growth color of the detection reaction:

(Number) colonies Salmon-Gal X-Gluc indole

1. good dark blue-violet + + +

2. good red +

3. good red +

4. good red +

5. good colorless. . .

6. no

7. no

8. no

9. no

10. no

11. no

12. good light blue-turquoise The examples above show that when using the nutrient media according to the invention, all combinations which can arise with regard to the galactosidase and glucuronidase activity can be differentiated:

a) Colonies of bacteria which have none of these enzyme activities remain undyed;

b) Colonies of bacteria that have both enzyme activities are colored dark blue-violet; this combination is characteristic of E. coli. The specificity of this detection can be further increased by the additional indole reaction.

c) Colonies of bacteria that have only galactosidase activity are colored red; this finding is characteristic of coliform bacteria.

d) Colonies of bacteria that have only glucuronidase activity are colored light blue-turquoise. This case occurs, for example, with some strains from the genus Salmonella.

Claims

Expectations

1. Nutrient medium for the detection of Escherichia coli and / or coliform bacteria consisting essentially of complex nutrient media, salts for adjusting pH and ionic strength, chromogenic substrates for β-galactosidase and for β-glucuronidase and possibly other conventional ones Ingredients, characterized gekenn¬ characterized in that two differently substituted indolyl derivatives are contained as chromogenic radical for the ß-galactosidase and ß-glucuronidase substrates.

2. Nutrient medium according to claim 1, characterized in that a gelling agent is additionally contained.

3. Nutrient medium according to one of claims 1 to 2, characterized gekenn¬ characterized in that 6-chloro-3-indolyl-ß-D-galactopyranoside as ß-galactosidase substrate and 5-bromo ^ 4-chloro as ß-glucuronidase substrate -3- indolyl-ß-D-glucuronide is included.

4. nutrient medium according to one of claims 1 to 3, characterized gekenn¬ characterized in that an enzyme inductor is included as an additional component.

5. Nutrient medium according to one of claims 1 to 4, characterized in that growth factors and / or

Growth inhibitors are included.

6. Dry preparation for the preparation of a nutrient medium according to one of claims 1 to 5.

7. Use of a nutrient medium according to one of claims 1 to 5 for the detection of Escherichia coli and / or coliform bacteria in a test sample.

8. A method for the detection of Escherichia coli and / or coliform bacteria, characterized by the following method steps: a) inoculating a nutrient medium according to one of claims 1 to 5 with an examination sample; b) incubate the inoculated nutrient medium until bacterial colonies become visible; c) Observation of the colored bacterial colonies.