WO1996007755A2

WO1996007755A2 - Method of performing confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria

Info

Publication number: WO1996007755A2
Application number: PCT/CZ1995/000020
Authority: WO
Inventors: Boz^¿ena SEDLINSKÁ; Jir^¿í HÄUSLER; Vlastimil S^¿TE^¿TINA
Original assignee: Lachema A.S
Priority date: 1994-09-09
Filing date: 1995-09-11
Publication date: 1996-03-14
Also published as: CZ282401B6; CZ219294A3; WO1996007755A3

Abstract

The invention relates to a method of performing confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria, based on that onto a moistened adsorption matrix saturated with a fluorochrome substrate there is transfered from a selective culture medium an incubated membrane filter with colonies of microorganisms which upon further cultivation for 1 to 4 hours at an optimal temperature for the microorganisms in question is exposed to U.V. rays of a wave length of 350 to 370 nm.

Description

Method of Performing Confirmatory Tests by Fluorochrome Substrates in Indicators of Fecal Contamination or Potentially Pathogenic Bacteria

Field of the Art

The invention relates to a method of confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria, the aim of which is the confirmation of the taxonomical categorisation of the microorganism and better determination of the detected bacterial contamination.

State of the Art

The hitherto used method of identification of microorganismus is based on the study of their morphological, ecological, cultivational, physiological, biochemical, and eventually other significant characteristics.

In the case of bacteria that are difficult to distinguish morphologically, emphasis is laid only on the specification of their physiological and biochemical characteristics, whilst it is necessary to work with pure cultures the preparation of which is usually difficult. This method has several shortcomings. It is time-consuming as it takes more than a week and so there is danger of delay in cases of hygienic defects. Moreover, it is very elaborate as it is necessary to carry out 70 or more tests on various culture media to identify one bacterial strain. Besides this it requires highly qualified and experienced staff. In routine practice it is not necessary to carry out an exact identification of each microbial culture in order to classify hygienic or other defects because it is sufficient to perform confirmatory tests with cultures isolated from selective culture media. On the basis of such results it is possible to classify reliably each microorganism into the appropriate taxonomical category (species, genus) or just into the group (e.g.coliform bacteria).

Simple biochemical reactions are usually used for such confirmatory tests. For the identification of microorganism with a higher degree of reliability it is necessary to perform several parallel tests.

The Embodiment of the Invention

The above mentioned shortcomings are overcome by this invention the subject matter of which is that an appropriate industrially absorbing matrix prepared in advance is saturated by a specific fluorochrome for each microorganism in question. The above matrix is stored dry in the dark and sealed in a plastic bag at temperature below ♦4° C. Prior to the use the matrix is placed in a Petri-dish, it is moistened and then a membrane filter with grown colonies is transferred on it and incubated for another 1 to 4 hours. The result of the test is evaluated after termination of the cultivation under 366-nm UV light. Presumptive colonies of the tested microorganism show a blue fluorescence.

Method according to this invention is universal because it facilitates the determination of presumptive colonies of a certain microorganism previously cultured on any type of selective medium and does not require preparation of a costly medium with a fluorochrome substrate. Matrices according to this invention are ready to use up to the date of expiration. The invention does not require any extraordinary laboratory equipment except a U.V.lamp. The main advantage of the method according to this invention is the direct quantification of the studied microorganism and the determination of a ratio of its colonies in relation to colonies of other taxonomic categories.

Examples

Example 1

Determination of the number of colonies of presumptive Escherichia coli in drinking water

In a sample of drinking water the number of colonies of thermotolerant coliform bacteria on "HFC" medium is determined by the membrane filter method. After the cultivation the number of lactose-positive colonies is determined. The membrane filter is then transferred on the absorption matrix, which is moistened by distilled water and contains fluorochrome substrate, i.e. 4-methyl-umbelliferyl- 3- -D-glucuronide, (MUG). This matrix is placed in a Petri dish of diameter 6 cm. The covered Petri-dish is placed for 4 hours in a thermostat with an incubation temperature of 37-l°C. After cultivation the cover of the Petri-dish is removed and in a darkened room under 366-nm light the total count of blue fluorescent colonies, i.e. presumptive Escherichia coli is determined.

Example 2

Determination of the ratio between the number of coliform bacterial colonies and the number of presumptive Escherichia coli.

The number of coliform bacterial colonies in a sample of drinking water is determined by the membrane filter method on Endo-agar plates. The number of lactose-positive colonies, i.e. coliform bacteria, is determined after cultivation and cytochrome oxiase test.

Afterwards the membrane filter is transferred onto the absorption matrix and further steps follow as in Example 1. Blue fluorescent colonies indicate the number of presumptive Escherichia coli . From the ratio of the number of coliforms and the number of presumptive Escherichia coli it is possible to deduce the source of bacterial contamination.

Example 3

Performing tests for coliforms in the determination of enterococci

Enterococci are determined by the membrane filter method, by cultivation on absorption matrices saturated with liquid SF medium. After cultivation the membrane filter is transferred to a new absorption matrix saturated with culture medium containing fluorochrome substrate, i.e. 4-methylumbelliferyl- & -D-glucuronide. Further steps follow Example 1. The total count of light-blue fluorescent colonies is determined after additional cultivation.

Claims

Claim

1. The method of performing confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria, characterized in that onto a moistened absorption matrix saturated with a fluorochrome substrate there is placed a membrane filter that has been incubated in selective culture medium and contains colonies of microorganisms and upon further incubation for 1 to 4 hours at optimal temperatures for the microorganisms involved is exposed to U.V. rays of a wave length of 350 to 370 nm.