Microbe growth detection

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism

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435 4, 435 34, 435 39, C12Q 102

Patent

active

047585099

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to the detection of microbial growth.
There is often a requirement in microbiology to detect the growth of micro-organisms. Examples of this are food spoilage detection and anti-microbial susceptibility testing. In the latter example, a culture is prepared of an unknown organism and small quantities of antibiotic are added to determine the concentration required to prevent growth.
It is clearly important to be able to detect the growth as quickly as possible and various methods for doing so have already been applied or proposed. For example, measurement of optical density works well where the culture medium is itself reasonably transparent. Other proposed techniques include measurement of the circular polarisation of transmitted radiation, measurement of the capacitance beween two plates placed in the medium and measurement of conductivity using two electrodes immersed in the medium.
The present invention seeks to provide a method capable of detection of microbial growth at an earlier stage than the methods previously proposed.
In accordance with a first aspect of the present invention, there is provided a method of detection of microbial growth which comprises preparing a culture of an organism, analysing a portion taken from the whole culture in a pyrolysis mass spectrometer, and monitoring the relative height of the peak in the mass spectrum corresponding to a mass of 60 daltons, an increased height of the said peak relative to the remainer of the spectrum being indicative of growth of the micro-organism.
In accordance with a second aspect of the present invention, there is provided a pyrolysis spectrometer for enabling the growth of micro-organisms to be detected which mass spectrometer is tuned or constructed to provide a first signal corresponding only to the abundance of ions present having a mass of 60 daltons and a second signal corresponding to the abundance of ions at one or more of the masses, the relative magnitude of the first and second signals being indicative of microbial growth.
The invention will now be described further, by way of example, with reference to the accompanying drawings in which;
FIG. 1a is a typical pyrolysis mass spectrum of a bacterial culture in the absence of growth, and
FIG. 1b is a similar culture to 1a after four hours of growth.
To detect microbial growth, a liquid culture is prepared in the usual manner. A typical medium may comprise 15 mls of blood diluted in 85 mls of brain-heart infusion. The broth is kept shaken at a constant 37.degree. C., whereupon any micro-organism present should grow. To assess antibiotic susceptibility of the growing organisms, multiple cultures are prepared and several dilutions of various antibiotics are added. For example, in the case of the suspected presence of Staphylococcus aureus, it may be necessary to test for sensitivity to penicillin, tetracycline and chloramphenicol at dilutions ranging from 1 to 100 .mu.g/ml.
After a time interval of, say, 4 hours, about 10 ul of the broth are extracted from each culture and analyised by means of a mass spectrometer. For example, the extracted broth from each flask is used to coat a ferromagnetic foil or wire to be used as sample carrier. Suitable apparatus for handling such sample carriers is described in detail in United Kingdom Patent Appln. Ser. No. 8315956 now Published Specification No. 2,141,230.
Typically a small foil of nickel/iron alloy is coated with the said culture and dried for 1 hour in a vacuum dessicator or by using a stream of hot air for about 1 minute. The foil is inserted in small glass tube which is then evacuated in the inlet system of the mass spectrometer. An r.f. induction heater outside the glass tube is used to heat the foil to its Curie point temperature. For a 50:50 nickel/iron alloy this corresponds to 510.degree. C. At this temperature, pyrolysis of the components of the culture occurs and the molecular weights of the products are analysed by means of the deflection or filter system of the mass spectrometer.
The resulting spectra

REFERENCES:
patent: 3891508 (1975-06-01), Merrick
Risby et al.-J. Physical Chem., vol. 80, (1976), pp. 2839-2845.
Anhalt et al.-Analytical Chemistry, vol. 47, (Feb. 1975) pp. 219-224.
Windig et al.-Chem. Abst., vol. 98, (1983), p. 13934f.
Wieten et al.-Chem. Abst., vol. 101, (1984), p. 3497g.

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