JPH0695928B2 - Gas chromatograph for microbial culture - Google Patents

Description

TECHNICAL FIELD The present invention relates to a gas chromatograph suitable for automatically analyzing metabolites of microorganisms cultured in an incubator.

(Prior Art) In the field of follow-up investigation of chemotherapeutic agents and infection routes, since identification of bacterial species is an important factor, various identification techniques have been developed. As one of the methods, a method of accommodating bacteria in an incubator and analyzing a terminal metabolite by gas chromatography has been proposed. According to this method, accurate information on the terminal metabolites can be obtained, and the bacterial species can be identified with high accuracy.

(Problems to be Solved by the Invention) However, due to the use of anoxic gas for transferring the sample to the analytical column, there is a problem that the identifiable bacterial species are limited to anaerobic species. is there.

The present invention has been made in view of such problems, and an object thereof is to provide a gas chromatograph for microbial culture capable of analyzing final metabolites of both aerobic and anaerobic bacteria. It is in.

(Means for Solving the Problem) In order to solve such a problem, in the present invention, a carbon dioxide gas means, a humidifying means, and a culture air supply means comprising a selectively operable deoxidizing means, The microorganism culture container, the analysis column, a detection means for detecting the components separated by the column, a flow path for supplying air from the culture air supply means to the microorganism culture container, and the culture air A flow path for supplying air from the supply means to the analytical column as a carrier gas through the microorganism culture container and a switching means for selecting the flow path are provided.

(Operation) Since the gas supplied to the microorganism culture container is used as the carrier gas to transfer the atmosphere in the microorganism culture container to the column, the final metabolite can be analyzed without inhibiting the growth of the microorganism.

(Example) Therefore, the details of the present invention will be described below based on an illustrated example.

FIG. 1 shows an embodiment of the present invention, in which reference numeral 1 denotes an incubator for culturing bacteria, in which a first tube 3 having one end immersed in a culture solution 2, A second tube 5 having one end communicating with the upper space 4 is provided,
Each tube 3, 5 is connected to a hexagonal switching valve 6. 9
Is a culture gas generation device, a molecular sieve tube 10, accommodating granular copper, oxygen removal tube 12 that can be heated to the oxidation temperature by the heater 11, soda lime tube 13, and humidification tube 14 is configured to be connected in series. , Is connected to the detector 17 via the three-way branch pipe 16 and to the six-way valve 6 via the resistance pipe 18.

In the figure, reference numeral 15 is an analytical column filled with an oxidation resistant packing, one end of which is connected to the hexagonal switching valve 6 and the other end of which is connected to the detector 17. In the figure, reference numeral 26 is a stop valve and reference numeral 28.
Indicates a hydrogen supply pipe for operating the detector 17.

In this embodiment, the culture solution 2 and aerobic bacteria are stored in the incubator 1, the six-way switching valve 6 is set to the position shown by the solid line in the figure, and the three-way switching valve is set to the position shown by the dotted line in the figure to further remove oxygen. Supply port with tube 12 kept at room temperature
When air is supplied from 25, the air will flow into the molecular sieve tube 10
Oxygen in the air is removed by the
After passing 12, the carbon dioxide gas is removed by the soda lime cylinder 13,
After being humidified in the humidifying cylinder 14, it is branched by a three-way branch pipe 16, one of which flows through a resistance pipe 18 into an analytical column 15 connected to the six-way switching valve 6. The bacteria in the culture solution continue to grow while consuming oxygen of the air existing in the upper space of the incubator 1 and the nutrients of the culture solution 2, and discharge the final metabolite. When the inside of the incubator 1 exceeds the atmospheric pressure, excess gas passes from the pipe 5 through the hexagonal switching valve 6 and the stop valve 26.
Is released from the air into the atmosphere, the pressure inside the incubator 1 is maintained at atmospheric pressure, and the growth of bacteria is maintained.

The gas discharged from the other port of the three-way branch pipe 16 is
Through to the detector 17.

When the six-way switching valve 6 has been switched to the flow path indicated by the dotted line in the figure after the predetermined time has elapsed, the gas flowing from the three-way branch pipe 16 flows into the incubator 1 through the six-way switching valve 6 and the culture solution 2 is aerated and the final metabolite in the culture solution 2 is expelled to the upper space 4 of the incubator,
It is discharged to the analytical column 15 through the tube 5. The analytical column 15 uses the air supplied through the incubator 1 as a carrier gas to perform the separation operation, and discharges the separated components to the detector 17. Thereby, the final metabolite of the bacterium in the incubator 1 can be known, and the bacterium can be identified. During the step of expelling the final metabolite, fresh gas is supplied into the incubator 1, and the bacteria can grow.

On the other hand, in the state in which anaerobic bacteria are stored in the incubator,
Air is supplied in a state where the heater 11 is operated to raise the temperature of the copper container to about 300 ° C. As a result, oxygen in the air is consumed to oxidize the copper in the oxygen removal cylinder and is removed from the air. Oxygen-depleted air is a soda lime tube
13, it is blown into the culture solution 2 in the incubator 1 through the humidifying cylinder 14. As a result, the anaerobic bacteria in the culture solution 2 continue to grow without being disturbed by the growth environment.

In this way, when the six-way switching valve 6 is switched to the flow path indicated by the dotted line in the figure after a predetermined time has elapsed, the oxygen-free air from the culture gas generator 9 passes through the culture solution 2. Gas in the headspace of the incubator is an analytical column 15
To discharge.

As a result, the final metabolite of the anaerobic bacterium is separated into each component and detected by the detector 17.

When investigating the number of bacteria at a specific point in time, set the switching valve 29 at the position shown by the solid line in the figure and the hexagonal switching valve 6 at the position shown by the solid line in the figure to prevent the growth of the bacteria. A gas to suppress may be injected.

[Example] Using 50 microliters of a 10% sucrose solution and 5 mg of saccharomyces cerevisace as a culture solution,
When ethanol was measured every 10 minutes while culturing the aerobic yeast at 20 ° C, it was found that the concentration of ethanol increases with the passage of time, that is, as the yeast grows, as shown in Fig. 2. It could be confirmed.

From this, it was revealed that the apparatus of the present invention can reliably measure the final metabolite of the aerobic bacterium without inhibiting the growth of the aerobic bacterium.

(Effects of the Invention) As described above, in the present invention, in the present invention, the decarbonation means, the humidification means, the culture air supply means composed of the selectively operable deoxidation means, the microorganism culture container, and the analysis A column, a detection means for detecting the components separated by the column, a flow path for supplying the air from the culture air supply means to the microorganism culture container, and the air from the culture air supply means via the microorganism culture container. Since it has a flow path for supplying as a carrier gas to the analytical column and a switching means for selecting these flow paths, the air used for culturing microorganisms can also be used as an analytical carrier gas, and can be used as a carrier gas source. Not only can the structure of the culture air supply source be covered by a single device to simplify the structure, but also anaerobic, while preventing unnecessary dilution of the sample and interference with microorganisms during analysis, Regardless of bacterial species such as aerobic, the final metabolite can be detected to identify the bacterial species.

[Brief description of drawings]

FIG. 1 is a block diagram of an apparatus showing one embodiment of the present invention, and FIG. 2 is a diagram showing measurement results in the process of culturing yeast according to the present invention. 1 ... Incubator, 2 ... Culture fluid 6 ... Switching valve, 9 ... Culture gas generator 10 ... Molecular sieve cylinder 11 ... Heater, 12 ... Oxygen removal cylinder 13 ... Soda lime cylinder 15 ... Analysis Column, 17 ... Detector

Claims (1)

[Claims] 1. A culture air supply means comprising a decarbonation means, a humidification means, and a selectively operable deoxygenation means,
The microorganism culture container, an analytical column, a detection means for detecting the components separated by the column, a flow path for supplying air from the culture air supply means to the microorganism culture container, and the culture air A gas chromatograph for culturing microorganisms, comprising a flow passage for supplying air from the supply means to the analytical column as a carrier gas through the microorganism culture container, and a switching means for selecting the flow passage.