CZ219294A3 - Method of making conformity tests by making use of fluorochrome substrata in indicators of sewage contamination or potentially pathogenic bacteria - Google Patents

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 confirmatory tests using fluorochrome substrates for faecal contamination indicators or potentially pathogenic bacteria.

Technical field

The invention relates to a method for performing confirmatory tests using fluorochrome substrates for faecal contamination indicators or potentially pathogenic bacteria, the purpose of which is to confirm the taxonomic category of microorganisms and thus to specify more precisely hygienically significant bacterial contamination.

BACKGROUND OF THE INVENTION

The current method of identification of microorganisms is based on monitoring their morphological, cultivation, physiological, biochemical, ecological, or other important properties.

In the case of bacteria which are morphologically difficult to discern or completely indistinguishable, their identification focuses only on the specification of their physiological and biochemical properties, while working with pure cultures of microorganisms, which are difficult to prepare.

This method of identification has a number of disadvantages. It is very lengthy, since it lasts more than a week, so there is a danger of delay in case of possible hygienic defects. In addition, it is labor intensive, since up to 70 or more tests on different culture media are required to accurately identify one bacterial strain, according to current scientific knowledge. It also requires highly qualified and well-trained staff.

For routine practice, it is not necessary to accurately identify individual cultures of microorganisms to classify hygiene or other defects, since it is fully sufficient to perform confirmatory tests with cultures isolated from selective media. Based on their results, microorganisms with high reliability can be assigned to the relevant taxonomic category (species, genus) or only to a certain group of microorganisms (eg coliforms ”).

Biochemical reactions are commonly used as confirmatory tests. To identify microorganisms, to achieve a higher degree of reliability, several tests should be performed side by side.

SUMMARY OF THE INVENTION

These drawbacks are solved by the process according to the invention, which consists in preparing an industrially suitable absorbent pad and saturating it with a specific fluorochrome substrate. The substrate thus prepared is stored in a dry state, sealed in an impermeable sheet, at a temperature of up to (less than) 4 ° C, in the absence of light.

Just prior to use, the plate is placed in a Petri dish, moistened, then sealed onto the membrane filter with colonies cultured on selective medium and cultured for 1 to 4 hours at a temperature that is optimal for the microorganism in question. The test result is read after cultivation under a UV source of 366 nm.

Presumptive colonies of the microorganism of interest show blue fluorescence.

The method of the invention is universal, since it allows the determination of presumptive colonies of a particular microorganism previously cultivated on any selective medium and does not require the immediate preparation of an economically expensive and laborious medium with a fluorochrome substrate.

Suggested pads may be available at any time until their expiration date.

In addition to the UV lamp, the method of the invention does not require any extra laboratory equipment.

The main advantage of the method according to the invention is the possibility of direct quantitative determination of the microorganism of interest and determination of the ratio of the number of its colonies to the number of colonies of other taxonomic categories.

Examples i

Example 1. i i

Determination of presumptive number of eacherichia coli colonies in drinking water.

In the drinking water sample, the number of colonies of thermotolerant coloform bacteria on the "m PC" medium is determined by the membrane filter method. After cultivation, ae is determined for the number of lactose-positive colonies.

The membrane filter is then transferred to a distilled water-moistened absorbent pad containing a fluorochrome substrate, i.e. 4-methylumbelliferyl-A-D-glucuronide (MUG), which is placed in a sterile 6 cm diameter Petri dish. After lidting, the Petri dish is placed for 4 hours in a thermostat, thermopapered to an incubation temperature of 37-1 ° C. After cultivation, the lid of the Petri dish is opened and the total number of light blue fluorescent colonies is determined in a darkened room with 366 nm UV light. i.e. presumptive Escherichia coli.

Example 2.

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

The number of colonies of coliform bacteria on Endo agar is determined by the membrane filter method in the drinking water sample. After culturing and performing the cytochrome oxidase assay, the number of lactosopositive colonies, i.e. coliforms, is determined. ·

The membrane filter is then transferred to an absorbent pad and proceeded further as in Example 1.

Blue fluorescing colonies indicate the number of presumptive Escherichia coli.

From the ratio of coliforms to presumptive counts

Escherichia coli can be inferred from the origin of bacterial contamination.

Example 3.

Performing a confirmatory test for the determination of enterococci.

Snterococci are determined by the method of membrane filters, which are cultured on absorbent pads saturated with liquid medium (SP medium). After cultivation, the membrane filter is transferred to a new absorbent pad saturated with a culture medium containing a fluorochrome substrate, i.e. 4-methyl-umbelliferyl-β-glucoside.

The procedure is as in Example 1.

After additional culture, the total number of light blue fluorescing colonies was determined.

Claims (1)

Patent claims Method for performing confirmatory tests using fluorochrome substrates for faecal contamination indicators or potentially pathogenic bacteria, characterized in that a moistened absorbent pad saturated with a fluorochrome substrate is applied from a selective medium a cultivated membrane filter with colonies of raicrogranisms, which after cultivation 1 to 4 hours at optimal temperature for microorganism, UV light irradiates with wavelength 350 to 370 nm.