JPH0440884A - Non-vapor phase, high-pressure microbial isolation system - Google Patents

Abstract

PURPOSE:To provide the title system intended to be freed from risks of explosion and secure safety, so designed that a culture chamber is filled with e.g. liquid paraffin as fluid and this fluid is ensured to circulate under constant pressure through a specific mechanism to make microbial inoculation and culture and withdrawal through suction. CONSTITUTION:The entire circuit comprising an injection circuit and a suction circuit each connected, via a closed circuit, to a culture equipment is filled with a fluid (e.g. liquid paraffin) immiscible with water in a pressurized state. This fluid can be circulated under constant pressure by using pump means each connected in series to both the circuits. Thence, the tip of a microbial inoculation tube 25 is first made to descend onto the agar-agar medium in a laboratory dish 17 in a culture chamber 11 and strain is injected into the medium. After incubation, the tip of a suction tube 26 is inserted into a microbi al colony followed by sucking up into a storage section 38 with a detachable collection part 8. Thus, use of such a fluid as liquid paraffin will free the pres ent system from risks of explosion etc. and greatly improve its safety.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to a microorganism isolation device for culturing and isolating microorganisms, and in particular, for cultivating microorganisms collected under high pressure under high pressure, such as those collected in the deep sea. The present invention relates to a high-pressure airless phase microorganism isolation device for isolating microorganisms.

[Prior Art] In order to isolate and culture hyperbaric deep-sea microorganisms that are killed by reduced pressure, a deep-sea microorganism isolation device that combines an incubator capable of maintaining high pressure and a constant temperature control device is used.

The incubator is connected to a pressurizing pump, pressurizing gas with the pressurizing pump to maintain a high-pressure environment, and controlling the temperature with a constant-temperature control device to create a high-pressure constant-temperature environment.

[Problem to be solved by the invention] The conventional deep-sea microorganism isolation device described above creates a desired high-pressure environment inside the incubator by pressurizing gas, but the high-pressure state due to gas has a large compression ratio and a large amount of heat. It is. Furthermore, if there is a malfunction of the equipment or an erroneous operation during inoculation or removal of microorganisms, there is a risk that the incubator and connecting pipes under high pressure may explode, which is very dangerous.

Therefore, a technical problem arises that must be solved in order to eliminate the danger of explosion and provide a high-pressure isolation device that ensures safety.The present invention aims to solve this problem. .

[Means for Solving the Problems] The present invention was proposed to achieve the above object, and provides a microorganism that pressurizes the inside of an incubator to culture and isolate hyperbaric microorganisms in a high-pressure constant temperature environment. In the isolation device, the culture chamber of the culture vessel is filled with a fluid immiscible with water, such as liquid paraffin, and the other ends of the inoculation tube and suction tube inserted into the culture chamber are respectively A closed injection circuit and a suction circuit are provided in the culture chamber, and a pump means such as a double-acting cylinder is connected in series to each of the injection circuit and the suction circuit so that the fluid can be circulated at equal pressure. A storage section for a microorganism sample is connected in line in a conduit between the suction tube and the pump means, and the storage section is maintained in a pressurized state by valves provided at both ends and is detachable, and is connected to the pump means of the injection circuit. It is an object of the present invention to provide an airless phase high-pressure microorganism isolation device characterized in that the accommodated microorganisms are inoculated into a culture chamber, cultured, and the microorganisms are taken out by suction into the storage section. be.

[Operation] The entire circuit of the injection circuit and the suction circuit connected to the incubator in a closed circuit is filled with a fluid immiscible with water, such as fluid paraffin, and is pressurized to a high pressure. Pump means such as double-acting cylinders are connected in series to each of the injection circuit and the suction circuit, and the pump means can circulate the fluid while keeping the pressure of the fluid in equilibrium.

The sample of microorganisms accommodated under pressure in the pump means of the injection circuit is transferred to the incubator and cultured while the pressurized state is maintained by the injection operation of the pump means. When removing the cultured microorganisms, the pump means provided in the suction circuit is operated to suck the microorganisms into the suction tube. The storage section provided in the conduit between the suction tube and the pump means is removable, and when the sucked microorganisms reach the storage section, the valves at both ends of the storage section are closed to maintain the pressurized state. It can be removed, stored or transported.

“Embodiment” An embodiment of the present invention will be described below in detail with reference to the accompanying drawings. In Figure 1, (1) is an airless phase high-pressure microorganism isolation device (hereinafter referred to as isolation device), (2) is a stand, (3) is a pressure gauge, and (4) is a television camera. be. or,
(5) is a suction-side double-acting cylinder used when extracting microorganisms, which will be described later.

Figure 2 shows the isolation device (1), which includes an incubator (7) removably fixed to a stand (2) by bolts (6), (6), and the sides of the incubator (7). Collection section installed in
8). The incubator (7) has a main body (
9) and an upper lid (10), and a turntable is provided in the culture chamber (11) of the main body (9). θ
By rotating the shaft (1 knob (10), the turntable (+21) can be rotated from the outside. (The cup is a petri dish placed on the turntable (■ Culture medium (contains days).

In addition, (the gull is a passageway that communicates the culture chamber (11) with the outside, and although not shown, a similar passageway is further provided to connect the injection circuit and suction circuit, which will be described later). There is.

A filtration jacket (A) is provided at the bottom of the main body (9) to circulate constant temperature water, and a constant temperature water circulation device (not shown) is connected to the connection part JI2 to control the inside of the culture chamber (11) at a constant temperature. do.

Said main body (9) Hehol H1) G! l) The upper M (In), which has been tightened in..., is opened with an observation window (through) in the center and a pressure-resistant glass (through) is fitted, as shown in Figures 2 and 3. The upper surface of the petri dish (r7) can be observed using an endoscope (24). Also, there is a TV camera (4) on the top of the endoscope.
) can also be attached for observation on a CRT. As shown in Fig. 2, an inoculation tube (c), suction tubes (4), (4), etc. are arranged around the observation window (to), and a petri dish (r) is placed around the observation window (to).
7) The agar medium (1) can be lowered to inoculate and inhale microorganisms under high pressure. As shown in FIG. 5, it is fixedly installed through the upper i (+o), and Q slide tubes are slidably fitted to the tip portion.

The slide tube) is connected to a rod (to) via a connecting member (a), and the rod (g) is made to protrude from the upper surface of the upper lid θ0). A bearing (1) that pivotally supports the upper part of the rod (G)
) is screwed into the upper part of the agar medium (1), and the bolt (a) is screwed in to lower the rod (to), and the agar medium (1) is interlocked with the slide tube. When the slide tube (A) is raised, the bolt (2) is rotated in the upward direction, and the rod (G) is raised by the internal pressure of the culture chamber θ1). In addition, as shown in Figure 4, a deployment rod (c) is attached to the tip of the inoculation tube (c), and a turntable (c) is rotated.
A seawater sample injected onto an agar medium (transfer) can be averaged out.

As shown in Fig. 5, the suction tubes (4), (1)... are equipped with a suction nozzle (b) in place of the deployment rod (c), and have different mounting angles (α0) (main). In the example, 7°, 9°, 11°, 13°), Petri dish (
Microbial colonies can be collected at any location on r71.

Figure 6 shows the collection section (8), with valves V4A, V2O, V4C, and V4O lined up above and below the four storage sections (g), (t), (b), and (to) formed of translucent tubes. V5
A, V2O, V5C, V2O are provided, and the upper valve ■
6 is connected in parallel. Said storage section (g) (1) 0 force (
A) is the connection part J4A, J4B, J4C provided above and below.
.

J4D, J5A, J5B, J5C, J51), it can be freely attached and detached from the collection part (8). Also, there is a valve v3 at the bottom end.

V3O, V3C, and V2O are provided, and the suction pipe (4
) (a) are connected to... respectively.

Figure 7 is a circuit diagram of the isolation device (1), where (G) is a pressure pump, (B) is a constant temperature control device, (4I) is a constant temperature chamber of the constant temperature control device, and (42) is an injection side complex. It is a moving cylinder. The injection side double acting cylinder (42) and the suction side double acting cylinder (5) have upper and lower ports (43), (44), (4
5) Attach needle valves (47) and (48) to (46), respectively.
), (49) and (50) are provided to seal the inside of the cylinder. Also, although not shown, the piston rod (51) (51) and the cylinder (52) (
52), and the piston handle (53) (
Pistons (54) (54) are rotated by rotating the pistons (53).
) is formed so that it can be moved slightly.

Next, the operation process of the device (1) will be explained with reference to FIG.

In preparation for sterilization, fill the isolation device (1) with fluid/X+ rough-in.

(1) Close all valves.

(2) Make sure that the inoculation tube (a) and suction tube (4) (1) are raised upward.

(3) Connection part J3. Remove the JIO and - Bolts (21) (21) G that secure the top cover (10)! I) Remove (21) and remove the top cover (10).

(4) Pour the agar medium into the Petri dish and place it on the turntable (■).

(5) Set the top cover (10) on the incubator body (9) and tighten with the bolts (21) (21) (2+) (21), and connect the connection part J3. Connect JIO.

(6) Fill the pressure pump (b) with fluid paraffin.

(7) Remove the suction side double-acting cylinder (5) from the connection parts Jl and J2, connect it to the pressure pump (to), and connect the cylinder (52) to the pressure pump (to).
) Inject fluid paraffin into the upper and lower compartments.

After confirming that the air has gone out, replace the needle valve (49H5
Close 0).

(8) After opening the valve Vl and pumping fluid paraffin with the pressurizing pump (b) and removing air from the connection, connect the boat (46) on the outlet side of the suction side double-acting cylinder (5) with the Connect the connection part.

(9) Valve v2. v7. Open the vB, open the cap (not shown) of the observation window (a), and remove the pressure pump (g).
Send fluid paraffin from the tank to remove air. After checking the air release, tighten the cap.

(10) Suction line “A system” Noharub V3A, V4A
, Open V5A and valve V6, send fluid paraffin with the pressure pump (b), and bleed air at the connection J2.

After confirming air bleeding, close valves V3A, V4A, and V5A.

(10 Suction line "B system JrC system JrD system" is also operated in the same manner as in operation procedure 00) to bleed air.

(After air bleeding from the suction line is completed at 0, close the valve ■6, and close the inlet side boat (45
) and connection part J2.

(1→ Open valve VIO, send rough-in fluid with pressurizing pump (b), bleed air at connection J8, then close valve VIO and attach a blind plug to connection J8.

(Close valves V1. V2. VB.

(1!1G Open valve V9.VII, send fluid paraffin with the pressure pump (G), bleed air at connection J9, close valve V7.V9.Vll, and connect J9
Attach the blank plug.

(+119 Remove connection J3. J6. J?.

(r7) Remove the bolts (6) (6) that fix the incubator (7) to the stand (2), remove the incubator (7), and use high-pressure steam in a sterilizer (not shown). Sterilize.

2: Preparation for culture (1) Fix the sterilized incubator (7) to the stand (2).

(2) Remove the blind plug from the connection part J8 and connect it to the outlet side port (44) of the injection side double-acting cylinder (42) containing the seawater sample.

(3) Remove the blind plug from the connection part J9 and connect it to the inlet side boat (43) of the injection side double acting cylinder (42).

(4) Connection part J3. J6. Connect J7.

(5) Open all valves v1 to V11, and open the needle valve (49H50) of the suction side double-acting cylinder (5).

(6) Pressurize the rough-in with fluid using the pressure pump (g),
Pressure is increased to 200 kg/cm' while monitoring with a pressure gauge (3).

(7) After the pressurization operation is completed, H/L/B Vl, V2. V
3A, V3B, V3C, V2O, V4A, V4B
, V4C, V2O, V5A, V5B, V5C, V
2O, VB.

■Close 7.

3: Culture (1) Set the endoscope into the observation window (A).

(2) Rotate the knob 01) of the inoculation tube (c) and
24), lower the tip of the inoculation tube (c) to a predetermined height above the petri dish (r7).

(3) Needle valve of injection side double-acting cylinder (42) (
47) When (4B) is opened, the inside of the cylinder (52) and the culture chamber (I +) become equal pressure, and the injection side double-acting cylinder (42
) to operate the piston handle (53) of the piston (5).
4), the seawater sample in the cylinder (52) is injected onto the agar medium (1) in the petri dish (r7).

(4) Valve V9. Close the VIO and close the needle valve (47048) of the injection side double-acting cylinder (42).

(5) Turn the knob (31) of the inoculation tube (G) and expand the deployment rod (
c) Lower the agar medium (above the surface of the plate).

(6) Rotate the turntable (zero rotation knob (lF9) to rotate the Petri dish (r7) and spread the injected seawater sample uniformly with the spreading rod (to).

(7) Turn knob 01) and pull up the inoculation tube (c).

(8) 1 for the connection part of the water jacket (G) of the incubator (7)
．． Connect the hose of the constant temperature control device (a) to J12.

(9) Set the temperature of the constant temperature control device (7) and start operation.

4: Sample extraction (1) Rotate the turntable (IQ) to rotate the petri dish (r?) and place the target microbial colony at the tip of the suction tube (1) of the suction line "A system". position.

(2) Suction line [-A system] Noharub V3A, V4A
, V5A and valve v2. Open v6 and turn knob 01
) to attach the suction nozzle of the suction tube (1) to the agar medium (
Insert into the sun.

(3) Rotate the piston solenoid (53) of the suction side m-motion cylinder (5) to lower the piston (54),
Suction up microbial colonies.

(4) Suck up the agar medium into the translucent tube part of the storage section (G).
Stop operation and close valves V3A, V4A, and V5A.

(5) Turn the knob 01) of the suction tube (4) to raise the suction tube (A).

(6) The suction line "B system JrC system JrD system" is also operated in the same manner as the operation steps (1) to (5) to isolate the target microorganism colony.

(7) After colony isolation, valve v2. Close v6.

(8) Needle valve (4) of suction side double acting cylinder (5)
9) Close (50).

Ku) Connections J4A, J4B, J4C, J4D,
Remove J5A, JOB, J5C, and J5D, and place them in the storage section (
g) Remove (1) @ (to).

5: Post-treatment (+) Open the valve V3A, release the pressure in the culture chamber (11), and close the valve V3A again.

(2) Install new storage parts (G) (To) (B) (To), and check valves V4A, V4O, V4C, V2O, V5A,
Close V2O, V5C, and V2O.

(3) Valve V8. Close V116 (4) Connection part J3. Remove JIO.

(5) Remove the upper lid 00) from the incubator main body (9), and after taking out the Petri dish (r7), attach the upper lid θ0) to the main body (9).

(6) Connect the connecting part O.

(7) Remove connection J8, and remove valve V1. V2. VI
Open J8 and use the pressure pump (A) to pump fluid paraffin in the opposite direction to the line of the inoculation tube (C) for cleaning.
Drain the liquid paraffin from.

After that, close the valve VIO and attach a blind plug to the connection J8.

(8) H/L/B V3A, V4A, V5A,
Open VB, send fluid paraffin with the pressure pump (g), clean the suction line "A system", and let fluid paraffin flow out from connection J3.

After that, close valves V3A, V4A, and V5A.

(9) The suction lines "B system JrC system" and "D system" are also cleaned in the same manner as in step (8) described above.

(10) After cleaning, close the valves Vl, V2. Close VB.

(11) Connect connection J3, connect connection J1. J2.
Remove J9 and install blind plugs on each.

(12 suction side and injection side double acting cylinder (s) (42)
Remove and store.

(l→ Stop the operation of the constant temperature control device (7).

The above is the operation process of the device [tO], but the number of suction lines, the shape of the suction tubes (1), (1), etc. should not be limited to the above-mentioned example. Further, the suction side and injection side double-acting cylinders (5) and (42) can be replaced with pumps of other configurations, and furthermore, the operating procedure should not be limited to the one embodiment described above.

This invention can be modified in various ways without departing from the spirit of the invention, and it goes without saying that this invention extends to those modifications.

[Effects of the Invention] As described in detail in the above embodiment, the present invention allows the steps from injection to isolation of a microbial sample to be performed while maintaining a high pressure without reducing the pressure. Furthermore, since a fluid such as paraffin is used instead of gas as the pressurizing means, dangers such as explosion are eliminated and safety is significantly improved.

[Brief explanation of drawings]

The figures show one embodiment of the present invention, in which Figure 1 is a front view of an airless phase high-pressure microorganism isolation device installed on a stand, Figure 2 is a partially cutaway side view of the same device, and Figure 3 is a top cover of the device. A plan view, FIG. 4 is a longitudinal sectional view taken along the line A-A in FIG. 3 showing the inoculation tube, FIG. 5 is a longitudinal sectional view taken along the line BB in FIG. 3 showing the suction tube, and FIG. FIG. 7, a partially cutaway side view, is a circuit diagram of the device. (1)・----Airless phase high-pressure microorganism isolation device (5)・
...--Suction side double-acting cylinder (7)--Incubator (8)--...Collection section (10)...
・・Top lid (■・・・・Turntable (2)・・・・Observation window (4)・・・・Suction tube 04)・−・・Suction nozzle (G)・− ... Pressure pump (42) --- Injection side double-acting cylinder V4A, V4O, V4C, V2O --- Valve V5A, V2O, V5C, V2O --- Valve J4A, J4B, J4C, J4D... Connection part J5^, J5B, J5C, J5D... Connection part (11)... Culture chamber (0... Petri dish (c). ...Inoculation tube (c) ...-- Deployment rod

Claims (1)

[Claims] In a microorganism isolation device that pressurizes the inside of an incubator to culture and isolate hyperbaric microorganisms in a high-pressure, constant-temperature environment, a fluid immiscible with water, such as paraffin, is introduced into the culture chamber of the incubator. The other ends of the filled and inserted inoculation tube and suction tube are returned to the culture chamber to provide a closed injection circuit and a suction circuit, and double-acting is provided in the injection circuit and the suction circuit, respectively. Pumping means such as cylinders are connected in series so that the fluid can be circulated at equal pressure, and a microorganism sample storage section is connected in series in a conduit between the suction pipe and the pumping means,
The storage section is kept in a pressurized state by valves provided at both ends and is detachable, and the microorganisms contained in the pump means of the injection circuit are inoculated into the culture chamber and cultured, and the microorganisms are sucked into the storage section. An airless phase high-pressure microorganism isolation device characterized in that it is configured such that it can be removed.