Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1986-02-20
1989-01-17
Warden, Robert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 29, 435 34, 435810, 436110, 436172, 436903, C12Q 112, C12Q 102, C12Q 104
Patent
active
047987880
DESCRIPTION:
BRIEF SUMMARY
This invention relates to processes and materials for carrying out microchemical and microbiological tests. In particular, it relates to chemical tests to detect the presence of small quantities of nitrite, and the application of such tests to detecting the production of nitrite by microorganisms in culture, e.g. by reduction of nitrate supplied in the culture medium.
A well established test for small quantities of nitrite is known as the Griess test. This test relies on treating an aqueous material suspected of containing nitrate with p-aminobenzenesulphonic acid in strong acid solution (e.g. 5 N acetic acid) and with alpha-dimethylaminoaphthalene, also in strong acid solution.
In the presence of nitrite the test develops a red dye colour, but no colour is given if the test is carried out with a nitrite-free sample.
This test is widely applied as part of a technique for the species identification and classification of bacteria by their cultural application in many clinical microbiology laboratories. The test can also be used to detect traces of nitrite in other samples, e.g. water samples to be analysed in public health laboratories.
As it stands, this test generally requires visual assessment of colour development, and it involves the use and handling of two aromatic amine reagents which are somewhat noxious and chemically closely related to known carcinogens.
The aim of this invention is to provide a nitrite test that can be assessed by fluorescence in the resulting test material. It is also the aim of the invention to provide tests that can give a fluorescent result in a form suited to automatic fluorometric assessment, especially for example by fluorometry that uses similar excitation and emission wavelengths as other and possibly hitherto chemically unrelated microbial tests, which can therefore be assessed simultaneously or in the same batch by similar or the same instrumentation. A further aim of the invention is to provide nitrite detection tests adapted to microbiological use for assessment of the nitrate-reducing capacity of microbial cultures, e.g. to assist in their identification, in which the use of noxious reagents to develop a colour result is minimised or avoided.
According to the present invention, nitrite is detected in a sample possibly containing small quantities of nitrite, especially for example an aqueous sample, such as for example a suspension or culture of microorganisms cultured in the presence of adequately nitrite-free nitrate for a period that allows reduction of nitrate, if possible, or a specimen of groundwater, by contacting the sample under acid conditions with a small quanity of an acid-stable fluorophor which has a content of amino groups, and in which the amino groups contribute substantially to the fluorescence, thereby to cause reaction between the amino groups of the fluorophor and any nitrite present under the acid conditions, and assessing the presence or quantity of nitrite by change in the fluorescence of the fluorophor as a result of the reaction.
A microbiological culture test process according to the invention, for detecting the presence of nitrate-reducing microorganisms is an inoculum sample, which comprises:
(a) culturing a microorganism to be tested in the presence of adequately nitrite-free nitrate, for a period sufficient to allow reduction of nitrate by any microorganism (if present) that has the capacity to reduce nitrate to nitrite (e.g. 1-6 hours);
(b) exposing the culture medium after culture to (diazotising) acid conditions in the presence of an amino-containing fluorophor thereby to cause diazotisation of the fluorophor to the extent of any nitrite present in the medium to form a diazonium derivative (or further reaction product thereof) which is substantially less fluorescent (or fluoresces at a substantially different wavelength) than said amino-containing fluorophor, and
(c) assessing the fluorescence of the culture medium as treated by step (b), thereby to show a reduction in fluorescence in the presence of a nitrate-reducing microorganism.
REFERENCES:
patent: 3785929 (1984-01-01), Kronish
patent: 4434235 (1984-02-01), Rabi et al.
Coppola et al., "Nitrite in Meat Products Determined by Fluorescence Quenching of p-Aminobenzoate Ion", Journal of the AOAC (vol. 59, No. 4, 1976), pp. 783-786.
Coppola et al., (1976) in Chemical Abstracts vol. 85, No. 17, p. 121919, Item No. 121908r.
Spiegel Jack
Warden Robert J.
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