DE4324392A1 - Culture medium for the simultaneous detection of coliform bacteria and Escherichia coli - Google Patents

Description

The invention relates to nutrient media for the simultaneous detection of coliform bacteria and E. coli, as well as the use of these media for Detection of coliform bacteria and / or Escherichia coli in sub suchungsproben.

The detection and determination of the bacterial count of E. coli is extremely important parameter for the assessment of the microbiological quality of Food and Water: The presence of E. coli is an indication of fecal contamination. The detection and determination of coli Bacteria as indicator germs are similarly important because their Presence on hygiene defects indicates. This results in the emergency in a sample, both the presence of E. coli and of detect coliform germs and determine the respective bacterial counts to be able to.

In addition to 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 introduced.

Thus, it was found that the detection of β-glucuronidase activity using chromogenic or fluorogenic substrates highly specific for E. coli is. For glucuronidase detection, FRAMPTON et al .; J. Food Prot. 51, 402-404 (1988) and WATKINS et al .; Appl. Environ. Micro biol. 54, 145-150 (1988) discloses the substrate 5-bromo-4-chloro-3-indolyl-β-D- glucuronide (X-GLUC). The blue reaction product (indigo) is easily recognizable on the nutrient medium and independent of the pH value Diffusion into the nutrient 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-β-D-galactopyranoside (X-GAL), that of MANAFI and KNEIFEL; Centralbl. Hyg. 189 225-234 (1989).

Others for the detection of β-glucuronidase activity and Galacto sidase activity suitable chromogenic and fluorogenic enzyme substrates are:

• 4-Nitrophenyl-β-D-glucuronide and 4-methylumbelliferyl-β-D-glucuronide (MUG)
  (Detection of β-glucuronidase activity)
• o-Nitrophenyl-β-D-galactopyranoside (ONPGAL), p-nitrophenyl-β-D-galactopyranoside (PNPGAL) and 4-methylumbelliferyl-β-D-galactopyranoside (MUGAL)
  (Evidence of galactosidase activity).

Thus, from mixed cultures without additional effort, both the coli Bacteria as well as E. coli should be characterized solid nutrient media at the same time the detection of glucuronidase in addition to the Allow galactosidase activity. From the substrates X-GAL and X-GLUC can be used only one, since the products of both Detection reactions are identical. Therefore, for example, was the Glucuronidase detection with X-GLUC with evidence of Lactose utilization by means of pH indicators combined (MATNER et al., J. Food Prot. 53, 145-150 (1990) and DARTORY and HOWARD; Lett. Appl. Microbiol. 15 (6), 273-276 (1992)). From other authors were the Galactosidase detection with X-gal and glucuronidase detection with the fluorogenic substrate MUG combined (MANAFI and KNEIFEL; Centralbl. Hyg. 189, 225-234 (1989).

In both cases, the detection of the second enzyme activity is due to the Unspecificity of the pH change or the difficulties in the fluorescence detection of methylumbelliferone (works in abge darkened room under UV light; Need to be the medium for the Neutralize proof with alkali; Quenching reactions in detection the fluorescence; strong diffusion of the fluorescent dye in the Nutrient medium) has considerable disadvantages.

So it turns the Task to provide improved nutrient media that are specific to the specific Detection of both glucuronidase activity and the Allow galactosidase activity on the same nutrient medium.

The registration number is CAS 13 38 182-21-5 6-Chloro-3-indolyl-β-D-galactopyranoside (Salmon-Gal / Rose-Gal). It has been found that this compound serves as a chromogenic substrate for Galactosidase can be used, in addition to chromogenic Substrate for glucuronidase 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC) can be used. The use of these two substrates allows a clear differentiation of the two enzyme activities the medium: galactosidase produces a red color, glucuronidase a turquoise-blue color; Bacterial colonies in which both enzyme activities are present, are dark violet colored. The colors are regardless of the pH, they easily diffuse on the nutrient medium do not fertilize, but remain in the colonies. The Stains remain stable for several days.

The invention relates to nutrient media for the detection of Escherichia coli and / or coliform bacteria consisting essentially from complex nutrient bases, salts for the adjustment of pH and Ionic strength, chromogenic substrates for β-galactosidase and for β-glucuronidase and, where appropriate, further customary constituents, characterized in that as chromogenic residue for the β-galactosidase and β-glucuronidase substrates two differently substituted indolyl derivatives are contained, wherein the nutrient medium additionally gelling can be added.  

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

The invention relates to the use of nutrient media with the above given composition 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 Steps:

• a) inoculating a nutrient medium with a test sample, wherein the nutrient medium has the composition according to the invention;
• b) incubating the inoculated nutrient medium until bacterial colonies become visible;
• c) Observation of the stained bacterial colonies.

The basic components contained in the nutrient media according to the invention are known in the art. These include gelling agents, for example Agar Agar. For certain applications, it is common to have a nutrient medium prepare without agar-agar and then with this a carrier, for. B. Nutrient carton to soak to create a solid breeding ground.

According to the invention, gelling agents are used in a broader sense Understood by means of which a nutrient solution in a solid or Semi-solid form is brought, and possibly also subsequently can be added. The skilled person are apart from the other mentioned known for this purpose suitable additives.

Furthermore, the nutrient media according to the invention contain complex nutrients soil components, such as yeast or meat extract, peptones from various those protein-containing products. Other components of the invention proper nutrient media are neutral salts for adjusting the ionic strength,  z. B. NaCl, as well as buffer salts for adjusting the pH, z. B. primary and secondary potassium or sodium phosphate. Preference is given to a pH set between 6 and 8.

Contain the chromogenic substrates used in the invention various substituted indole derivatives as chromogenic radicals, each in glycosidic linkage to galactopyranose or glucuron, respectively acid bound. The indole derivatives are chosen so that the resulting Indigoderivate have different colors. By the enzymatic cleavage thus results in two differently substituted ones Indoxyl derivatives, each under the action of atmospheric oxygen to dimerize differently colored indigo derivatives oxidatively. A suitable Combination of two such dyes is exemplified. However, they are from the chemistry of the indigo precursors further Indoxylderivate known, the react differently colored indigo dyes, and in similar Way can be used for the purpose of the invention. So Indoxyl and 5,7-dibromoindoxyl give a blue, 6-bromoindoxyl purple indigo derivative.

To get into the bacteria the expression of the enzymes on which the proof Reactions are based to stimulate enzyme inducers are added; so For example, isopropyl-β-D-1-thiogalactopyranoside (IPTG) increases the Galactosidase activity and methyl-β-D-glucuronide the glucuronidase Activity.

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

• - Growth factors supplement the nutrient media, these include For example, organic compounds such as pyruvic acid (Pyruvate) or L-tryptophan. An addition of L-tryptophan made possible it, the specific for E. coli indole detection in addition to the Detection of glucuronidase and galactosidase activity perform.  
• - Many test samples contain unwanted accompanying germs, their growth is reduced by growth inhibitors can. For this purpose, those skilled in the art, for example, Deter such as bile salts, lauryl sulfate or Tergitol®, as well as the more potent novobiocin known. Novobiocin-containing nutrient media suppress the accompanying flora very strongly; the inhibitory effect of other mentioned inhibitors is lower. Nutrient media, the Novobiocin or similarly strong inhibitors are included particularly suitable for studies on samples that are strongly associated with Accompanying germs are contaminated. For this purpose are preferred Lauryl sulfate and novobiocin combined. In contrast, nutrient media, containing only the weaker inhibitors, especially suitable for samples with sublethal damaged germs. Preferred for this purpose is the use of lauryl sulfate.

Commercially, the nutrient media are often used as dry preparations (Nutrient media) provided with water, dissolved and sterilized. Such products and their application are the Specialist familiar. In a preferred embodiment, the solid components of the nutrient media according to the invention to a dry powder mixed together and optionally additionally granulated. These Processing steps are familiar to the person skilled in the art.

Nutrient media, which were initially prepared without the addition of a gelling agent For example, after sterilization, they may serve as sterile ones To impregnate cardboard discs, then filter media for incubation be hung up. Such methods are also known from the prior Technique known.

The occurrence of E. coli is considered as an indication of fecal contamination the sample; Coliform bacteria are considered an indication of hygiene deficiencies. It can be examined liquid or solid samples:

• - Liquid samples, such as wells, surface, drinking water or drinks, are applied directly to the nutrient medium. If the sample contains a very high bacterial count, it is prediluted with sterile saline or sterile peptone water. In general, 100 μl of liquid inoculum are applied and distributed on the nutrient medium.
  If the sample contains only a few germs, it can be enriched by centrifugation before application: a larger volume, for example a few milliliters, are centrifuged and the bacteria-containing 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, e.g. As drinking water, is the filtration. In this case, a larger volume of the sample is sucked by means of negative pressure through a membrane filter; To this end, membrane filters made of cellulose nitrate with a pore size of 0.45 μm are common. Then the filter is placed on the nutrient medium and incubated.
• - Solid samples can be streaked directly on the nutrient medium or rinsed with sterile saline or with sterile peptone water or homogenized, then the suspension is applied to the nutrient medium.
  For example, solid samples may be mixed together with ten parts of buffered peptone water. These devices can be used, which are known under the name "stomacher". Subsequently, the suspension thus obtained or a dilution thereof is applied to the nutrient medium.

The details of such methods and possible variants of the method are familiar to the expert and are in manuals, such as. B. Compendium of Methods for the Microbiological Examination of Foods (M.L. Bacon (ed.); (1984) American Public Health Association), described.  

After inoculation, the nutrient media are incubated, with the Incubation temperature is typically between 25 ° C and 50 ° C. The incubation period is typically between 12 and 48 hours. The incubation conditions, the expert varies, for example, in Dependence on the examined sample. Preferred are the following Incubation conditions: The inoculated nutrient media are aerobic Conditions incubated at 35-37 ° C for 24 hours. With little out marked reaction or to hedge a negative result Increase the incubation period by 24 hours. In general are however, the results can be evaluated after 24 hours of incubation.

According to the invention are nutrient media with the following ingredients and Concentration ranges preferred: meat extract (2-5 g / l), NaCl (1-10 g / l), sodium pyruvate (0.5-2 g / l), potassium dihydrogen 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 novobiocin (Sodium salt, 0.003-0.01 g / L). Particularly preferred nutrient medium Compositions are described in Examples 1 and 2.

The composition of the culture media according to the invention allows all active ingredients to a powder mixture or granules mix together. This mixture can be dissolved directly in water and be autoclaved.

The use of nutrient media with a combination according to the invention Usually, after 24 hours, the results are already allowed even if the bacteria are sublethally damaged. In contrast, over must in known methods, eg. Using EMX agar, Incubated for 48 hours (Manafi, M. and W. Kneifel (1989) Zentralbl. Hyg. 189, 225-234).

Examples

The following examples are intended to explain the subject matter in more detail no limitation of the subject invention Specialist known general working methods not explained in detail; Manuals, such as Compendium of Methods for the Microbiological Examination of Foods (M.L. Bacon (ed.); (1984) American Public Health Association) describe such methods.

example 1 Nutrient medium with low inhibitory activity against gram positive bacteria

component Quantity (g / l) meat extract 3.0 NaCl 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 demineralized together in one liter Dissolved water and the nutrient medium autoclaved (15 minutes at 121 ° C). The nutrient medium has a pH of 6.8 ± 0.2, if necessary, the Adjust pH with sterile filtered sodium hydroxide or hydrochloric acid. Subsequently, the nutrient medium is cooled in a water bath to 45 ° C. and in a known manner z. B. poured into Petri dishes. The plates of this Nutrient media are clear and colorless.

This medium also allows for a very high recovery rate sublethal damaged bacteria.  

Example 2 Nutrient medium with increased inhibitory activity against gram positive bacteria

component Quantity (g / l) meat extract 3.0 NaCl 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 use E. coli or coliform bacteria even in the presence of a strong accompanying flora.  

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

bacterial strains 1. Escherichia coli ATCC 25922 2. Citrobacter freundii 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 subtilis ATCC 6633 9. Enterococcus faecalis ATCC 19433 10. Enterococcus faecium ATCC 6569 11. Listeria monocytogenes ATCC 19118 12. Salmonella sp. Serotype anatum ATCC 9270

In each case a Impföse of bacteria from a preculture of the respective Strains 1-12 are suspended in 5 ml of sterile peptone water. Each 100 μl of each suspension are placed on each Petri dish, the Nutrient medium according to Example 1 contains, dripped and distributed. The Inoculated nutrient media are used under aerobic conditions at 35 ° C for Incubated for 24 hours. Subsequently, growth, color of the colonies and the result of the detection reactions with Salmon-Gal and X-GLUC evaluated and carried out the indole reaction. The following results were found:

Example 4 Culture of different bacteria on a nutrient medium according to example 2

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

The above examples show that when using the Nutrient media according to the invention all combinations that relate 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 dark blue-violet colored; this combination is for E. coli characte teristic. The specificity of this proof can be determined by the additional further increase indole reaction.
• c) colonies of bacteria having only galactosidase activity, are colored red; This finding is characteristic of coliform bacteria teristic.
• d) colonies of bacteria having only glucuronidase activity, are light blue-turquoise colored. For example, this case occurs with some Tribes of the genus Salmonella on.

Claims (8)

1. Nutrient medium for the detection of Escherichia coli and / or coliform bacteria consisting essentially of complex nutrient bases, salts for adjusting pH and ionic strength, chromogenic substrates for β-galactosidase and for β-glucuronidase and optionally further customary components, characterized that two differently substituted indolyl derivatives are contained as the chromogenic residue for the β-galactosidase and β-glucuronidase substrates. 2. Nursing medium according to claim 1, characterized in that additional Lich a gelling agent is included. 3. Nursing medium according to one of claims 1 to 2, characterized marked indicates that the β-galactosidase substrate is 6-chloro-3-indolyl-β-D- galactopyranoside and as β-glucuronidase substrate 5-bromo-4-chloro-3- Indolyl-β-D-glucuronide is included. 4. Nursing medium according to one of claims 1 to 3, characterized marked records that contain an enzyme inducer as an additional component is. 5. Nursing medium according to one of claims 1 to 4, characterized marked records that as additional components growth factors and / or growth inhibitors are included. 6. Dry preparation for the preparation of a nutrient medium after a 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 any one of claims 1 to 5 with a test sample;
• b) incubating the inoculated nutrient medium until bacterial colonies become visible;
• c) Observation of the stained bacterial colonies.