JP3549949B2 - Low oxygen incubator - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to an improvement of an incubator for cells of microorganisms or animals and plants.
[0002]
[Prior art]
The growth of microorganisms and the culture of animals and plants are influenced by various factors such as temperature, gas concentration and composition of the culture chamber, culture solution, medium pH, and the like, depending on the object, and the growth is changed.
Particularly, the medium, the medium pH, the O ₂ concentration and the CO ₂ concentration in the culture chamber are important.
The CO ₂ concentration is important from the viewpoint of keeping the pH of the medium constant, and the concentration control of O ₂ is important from the aerobic and anaerobic properties of microorganisms and the type of cells. Until now keeping the culture chamber in a constant temperature, there is a structure to take into hot water through the wall so that the temperature change is reduced, also the medium the concentration of CO ₂ so that the pH is less likely change with a buffer containing sodium bicarbonate Is increasing. In another example, a CO ₂ introduction port and an N ₂ introduction port are provided, and the gas in the CO ₂ cylinder or the N ₂ cylinder is decompressed by a pressure reducing valve and connected by a pipe for introduction. Then, the concentration of O ₂ and CO _{2 in the} culture chamber is measured, and when the concentration deviates from a specified value, the gas concentration in the culture chamber is maintained constant while introducing these gases manually or automatically from the pipe. Culture experiments are ongoing. However, culturing takes a long time, and during that time, if the concentration of each gas changes due to the exhaustion of the gas in the cylinder, etc., the experiment often fails.
[0003]
Although the consumption amounts of N ₂ and CO ₂ vary depending on the set concentrations, N ₂ is generally larger. The experimenter has extra troubles, such as having to replace a heavy cylinder if the cylinder is lost while paying attention to the remaining amount.
When the culture is temporarily observed by opening the door in the course of the experiment, the surrounding air flows simultaneously with the opening of the door, the O ₂ concentration rises to 21%, and the CO ₂ concentration rises to 0.03%. Go down. When the door is closed, a large amount of N ₂ and CO ₂ needs to flow in, return to the original environment in a short time, and minimize the effect on the culture.
[0004]
[Problems to be solved by the invention]
Effort researchers automatically collect supplied to N ₂ in the atmosphere in order to minimize, O ₂ concentration to automatically constant (provided that at most 21%) as the, the N ₂ gas cylinder Eliminate exchanges. However, since CO ₂ is not naturally present in a large amount, it is necessary to bring a separately generated gas, so a CO ₂ cylinder is added separately.
[0005]
[Means for Solving the Problems]
An airtight culture chamber having a hot water jacket 2 on the wall surface and a door 3 on the front side, wherein the culture chamber is provided with an inlet for introducing N ₂ and CO ₂ and a check valve 25 having a constant sealing pressure. a closing sensor for detecting the open or closed state of the O ₂ sensors and / or CO ₂ sensor and door for detecting the outlet and the indoor gas concentration, the switching signal the O ₂ concentration and / or CO ₂ concentration is converted into an electrical signal To the control unit 29.
CO ₂ inlet of the culture chamber is a pipe connected to the CO ₂ pressure was adjusted using a pressure regulating valve from the CO ₂ source (bomb) via the automatic valve V ₃ 19 is supplied to the N ₂ inlet is An N ₂ supply is piped via an automatic valve V ₂ 20. The N ₂ supply sources are an N ₂ concentrator for automatically concentrating N ₂ in the air and a N ₂ storage tank, a membrane type for concentrating by an N ₂ permeable membrane and activated carbon of carbon molecular sieves (CMS). There is a PSA-type concentrator using a molecular sieve (known technology). N ₂ concentrated by the concentration device such as this is connected to the storage tank 23 of the N ₂ through the automatic valve V ₁ 21, after stored to the automatic valves in the N ₂ supply port of the culture chamber by a pipe Connected via V ₂ 20. Since the pressure sensor 18 is installed in the N ₂ storage tank 23, the gas pressure is measured, and this pressure signal is converted into an electric signal and transmitted to the control unit 29. N ₂ concentrator opens the automatic valve V ₁ after the lapse or a predetermined time after start the compressor from the controller 29 N ₂ concentration reaches a certain concentration when the pressure of the storage tank has dropped to a predetermined value P _L Supply gas to the storage tank. And stopped when the pressure of the storage tank rises to a predetermined value P _H. Automatic valve V ₂ 20 for supplying N ₂ gas from the storage tank to the culture chamber of the N ₂ is the O ₂ concentration of the culture chamber, the control unit controls to open and close by a signal a certain value or more, the O ₂ concentration Is maintained at a target constant value. That is, when the automatic valve V ₂ 20 is opened, it is when the O ₂ concentration in the culture chamber has reached a certain value or more, and when it is closed, it is when the concentration has reached the set value.
[0006]
Thus the O ₂ concentration when rises becomes automatic valve V ₂ 20 is opened N ₂ is filled, thereby the O ₂ concentration is reduced. However, surplus gas is discharged to the atmosphere from an air discharge port provided with a check valve having an operating pressure of about 0.1 kgf / cm ² · G so that the internal pressure of the culture chamber does not increase. Also, when the CO ₂ concentration is lowered to introduce CO ₂ gas from the bomb control unit is one of the CO ₂ source by the signal automatic valve V ₃ 19 is opened. Automatic valve _V 3 19 by the CO ₂ sensor signal and CO ₂ reaches the set value control unit 29 is stopped the introduction of CO ₂ becomes closed. The control unit is provided with a setter for setting the CO ₂ and O ₂ concentrations in the culture chamber to target values and a setter for setting the temperature to the target values. However, the temperature is not the main subject of the present invention and will not be described. The control of automatic valves V _3, V _2, the control unit 29 so that becomes the target value set by the setter O ₂ sensors and / or CO ₂ the automatic valve V ₃ while monitoring the sensor detection _signal, V performing a _second opening and closing control, but is when the difference between the target value and the detected signal is large continuous open state, controlled to be the valve approaches within a certain reference value to the target value while repeating the opening and closing at predetermined time intervals I do.
[0007]
【Example】
FIG. 1 shows a flowchart of a preferred embodiment of the present invention.
There is an airtight culture chamber 1 having a hot water jacket 2 and a door 3 on the front surface in the wall. The door 3 can be opened and closed by a hinge 4. The open / closed state is detected based on whether or not the button 6 of the open / close sensor 5 is pressed, and the signal is transmitted to the control unit 29 via the signal line 7. The culture chamber 1 has conduits 27 and 28 for introducing N ₂ and CO ₂ , and a check valve 25 having a constant sealing pressure to prevent the internal pressure of the culture chamber from becoming too high. An outlet is provided.
The culture chamber 1 has an O ₂ sensor 9 and a CO ₂ sensor 9 ′ for detecting an atmospheric gas concentration, particularly, an O ₂ and CO ₂ concentration. This density signal is converted into an electric signal and transmitted to the control unit 29.
[0008]
The conduit 28 for introducing a CO ₂ CO ₂ pressure regulated to a low pressure by using a pressure regulating valve the pressure is connected CO ₂ cylinder 22 through an automatic valve _V 3 19 is supplied. _{N 2} supply source is connected by piping via a automatic valve _V 2 20 The N ₂ inlet tube 27. N ₂ source is concentrated to 99.9 to 99.99% of the concentration of _{N 2,} which is _{N 2} concentrator 24 (concentration of PSA type which automatically concentrated _{N 2} at concentrations containing argon in air it is possible) and a storage tank 23 of the _{N 2, N 2} gas produced concentrated by _{N 2} concentrator 24 via the automatic valve _V 1 21, after which stores connected to the storage tank 23 of the _{N 2, N 2} is connected to be introduced into the culture chamber 1 via the automatic valve _V 2 20 in _{N 2} conduit 27. N ₂ concentrator has a rectifying section and the compressed air supply unit, the concentrator is activated, the nitrogen gas fed into the compressed incorporated atmospheric nitrogen-enriched portion is concentrated by first compressed air supply However, a certain period of time is required until the device is activated and reaches a predetermined high concentration, and the concentration is low at the beginning of activation. In particular, it takes several minutes in the case of a PSA type concentrator. After the N ₂ concentration to not sent Therefore the initial low concentration of nitrogen gas in the storage tank 23 of the N ₂ through the fixed time after concentrator activated, the control unit 29 to the automatic valve V ₁ 21 to open controls ing. Also, it has been found that the concentration of the N ₂ concentrator decreases with an increase in the amount of gas taken out with respect to the supply air amount in both the membrane type and the PSA type. In order to keep the concentration in the culture room constant, the concentration of nitrogen gas obtained from the N ₂ concentrator must be higher than the target concentration (this is a low concentration in terms of oxygen concentration). Although initial start enrichment gas of the aforementioned N ₂ concentrator is achieved by opening the automatic valve V ₁ 21 after reaching a high concentration through a certain time we not only this, the automatic valve V ₁ 21 Open when you, by from N ₂ concentrator to the storage tank of the N ₂ that is a low pressure, is temporarily large amount N ₂ flows, temporary reduction in the concentration of enriched gas is generated, the concentration of the culture chamber Was disturbed. For this reason, a flow rate restricting means 30 is provided between the N ₂ concentrator and the N ₂ storage tank, and a constant flow rate valve that flows only a constant flow rate even if a pressure difference between the two flows, or between the two. It has been found that it is important to provide a flow restricting means (orifice or the like) that allows only a predetermined flow rate at which the concentration of the gas supplied from the N ₂ concentrator can be obtained at a target concentration even when a pressure difference occurs. This flow restricting means 30 is provided in a piping path connecting the N ₂ concentrator 24 and the N ₂ storage tank 23.
[0009]
When the reservoir tank 23 in which N ₂ door of the culture chamber has been opened and closed, rapidly N ₂ concentrator as a large amount of gas to recover steady-concentration can be supplied in a short period of time of at most a small N ₂ is filled with zeolite capable of adsorbing and holding N ₂ so that N ₂ can be stored. Three times or more N ₂ can stored for the same pressure in a volume of normal tank by way. However, good results cannot be obtained unless the zeolite is combined with a low water content gas generated by an N ₂ concentrator that does not contain water, because the ability of the zeolite to absorb and retain N ₂ is deteriorated by water. Both a membrane type concentrator and a concentrated nitrogen gas obtained by a PSA type concentrator using CMS can provide a dry gas with low moisture. A pressure sensor 18 is attached to the N ₂ storage tank 23, and the pressure of the gas is measured and transmitted to the control unit 29 via the signal line 17. The pressure decreases as the volume of the stored gas decreases, and indicates higher pressure as the volume of the stored gas increases. When the O ₂ concentration in the culture chamber becomes higher than the set value and becomes higher than the set value, the control unit 29 opens the automatic valve V ₂ 20 according to the signal and supplies N ₂ , so that the gas in the tank is consumed. N ₂ concentrator 24 of PSA expression detects that the pressure of the storage tank 23 has fallen to a predetermined value P _L by the pressure sensor 18, which through a signal line 17 and transmitted to the control unit 29 control unit 29 signal line 16 to start the _{N 2} concentrator 24 by.
[0010]
In some cases, the culture is removed from the culture chamber during the course of the culture research, followed by research observation on the way, and then stored again to continue the culture. In such a case, if the door is opened and taken out, the outside air enters the double container, and the environment where the O2 concentration and CO2 concentration were controlled collapses, so immediately return to the original environment after the door is closed There is a need. For this reason, the situation where the door is opened and subsequently closed is detected by the open / close sensor 5, transmitted to the control unit 29 via the signal line 7, the N2 concentrator 24 is activated, and the automatic valve V1 is directly opened. Immediately after opening and closing the door, there is a large change in concentration, so a large amount of N2 is required, so it is necessary to generate and supply from the PSA N2 concentrator following the release of gas in the storage tank 23. . After the O2 concentration has decreased to a certain value, increasing the CO2 gas concentration lowers the O2 concentration, so that the CO2 consumption can be reduced as compared with the method of simultaneously supplying N2 gas and simultaneously supplying CO2. However, the length of time during which the environmental conditions are disturbed due to the nature of the culture object and fluctuations in the pH of the culture solution, etc., varies depending on the individual, so some N2 and CO2 must be supplied simultaneously. is there.
[0011]
When the automatic valve V ₁ 21 is closed is when the pressure in the storage tank rises to a predetermined value P _H, O ₂ concentration in the room the culture than the set value when the automatic valve V ₂ 20 is opened , And when it closes, the density has reached the set value. As described above, when the O ₂ concentration in the culture chamber 1 detected by the O ₂ sensor 9 or the CO ₂ sensor 9 ′ increases, the automatic valve V ₂ 20 is opened by the control unit 29 and N ₂ is discharged from the storage tank 23. As a result, the O ₂ concentration in the culture chamber 1 decreases. However, a surplus gas is discharged from the atmosphere discharge port through a conduit 26 provided with a check valve 25 so that the internal pressure of the culture chamber 1 does not increase. The CO ₂ concentration _{O 2} sensor 9 and CO ₂ sensor 9 'and transmitted to the control unit 29 by a signal line 11 for transmitting a reduction is detected CO ₂ concentration by the controller 29 to the automatic valve _V 3 19 by a signal line 13 of CO ₂ CO ₂ cylinder 22 and discharged into the culture chamber 1 in the open, achieving an increase in CO ₂ concentration. O ₂ sensor 9, CO ₂ sensor 9 'by CO ₂ concentration control unit 29 to be detected to reach a set value to stop the introduction of the CO ₂ automatic valve _V 3 19 is closed. The control unit 29 has a setting device for setting the CO ₂ concentration and the O ₂ concentration in the culture chamber to target values. Further, the temperature of the hot water jacket 2 is controlled by turning on and off the heater 8 by the electric wire 12 from the control unit 29 and set to the target temperature. The setting device for inputting the target value for achieving the target set value is also shown. It is omitted in the figure.
[0012]
【The invention's effect】
By carrying out the present invention, as a source of the incubator of N ₂ in the low-oxygen atmosphere, since the N ₂ with about 79% in the atmosphere can be used those concentrated above 99%, N ₂ gas cylinder or the like finite There is no need to worry about running out of gas like a supply source. While more volume the apparent small, the storage tank filled with an adsorbent to adsorb N _2, since the multiple of N ₂ appearance capacity can be stored, it is possible to supply a large amount of N ₂ to a temporary I can do it.
[Brief description of the drawings]
FIG. 1 is a flowchart of a preferred embodiment of the present invention.
[Explanation of symbols]
1 incubator 2 hot water jacket 3 door 4 hinged 5 closing sensor 6 button 8 heater 9 _{O 2} sensor 9 'CO ₂ sensor 7,10,11,13,17 signal line 12 wire 18 a pressure sensor 19 the automatic valve _{V 3}
20 automatic valve _{V 2}
21 Automatic valve V ₁
22 CO ₂ cylinder 23 Storage tank 24 N ₂ concentrator 25 Check valve 26,27,28 Conduit 29 Control unit 30 Flow rate limiting means

Claims (6)

The airtight culture room with a door has an N2 inlet, a CO2 inlet, and an O2 sensor that detects the gas concentration in the culture room.The detected concentration signal is transmitted to the control unit by an electric signal, and is sent to the N2 inlet. Is connected to the N2 supply source via the automatic valve V2, and the CO2 inlet is connected to the CO2 supply source via the automatic valve V3 via a pipe.The control unit controls the automatic valves V2 and V3 based on the signals from the O2 sensor and the CO2 sensor. N2 and CO2 are introduced and N2 and CO2 are introduced to keep the gas concentration in the culture chamber at the target constant concentration.In a low oxygen incubator, the N2 supply source is a N2 concentrator that automatically concentrates N2 in air and a storage. It is composed of a tank and N2 concentrated by an N2 concentrator is connected to the storage tank via an automatic valve V1 and stored.A pressure sensor is installed in the storage tank, and the stored N2 pressure is measured and controlled. The storage unit pressure is reduced to a certain value PL by the control unit, the N2 concentration After the elapse of a certain time, the automatic valve V1 is opened to supply N2 to the storage tank, and when the storage tank pressure rises to a certain value Pm, the automatic valve V1 is closed and the N2 concentrator is started. A hypoxic incubator, which is stopped to use N2 as the N2 supply source. 2. The low oxygen incubator according to claim 1, wherein the N2 storage tank is filled with zeolite that adsorbs N2. An open / close sensor that detects the open / closed state of the door is provided so that, in addition to the case where the N2 concentrator is started up to a certain pressure PL of the N2 storage tank, the door is opened and closed to start up and N2 enrichment is started. 3. The hypoxic incubator according to claim 1, wherein the incubator is configured as follows. 4. The low oxygen incubator according to claim 1, wherein when the N2 concentrator is started by detecting opening and closing of the door, the automatic valve V1 is controlled to be opened immediately after the N2 concentration is started. 5. The hypoxic incubator according to claim 1, wherein a flow restricting means is provided on a connection line between the N2 concentrator and the N2 storage tank. 6. The hypoxic incubator according to claim 1, wherein a constant flow valve is provided on a connection line between the N2 concentrator and the N2 storage tank.

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