JP4605559B2 - How to observe bacterial activity - Google Patents

Description

  The present invention relates to a technique for observing physiological activities of various bacteria living in the same place as benthic organisms in sediments on the seabed.

  The growth and activity of bacteria inhabiting marine sediments are affected by the presence of benthic organisms (benthos). In particular, when the agitation and modification action of sediments by benthic organisms is strong, the effect is also great. When observing or quantitatively grasping the growth and metabolic activity of bacteria in such sediments, it has been necessary to take a method of collecting a part of the sediment as a sample and analyzing it. is there. Therefore, in these processes, the operation becomes complicated, and it is difficult to increase the time / spatial resolution.

  On the other hand, as a conventional technique related to the present invention, a method for detecting microorganisms using an oxidation-reduction indicator has been proposed (see, for example, Patent Document 1).

JP-A-9-201198

  Bacteria and microorganisms can be detected by using the “improved method for the growth and non-color detection of bacteria, yeast, fungi, or cocci” described in Patent Document 1, but this method is only for pure culture of bacteria and microorganisms. Or it is a detection method effective only when culture | cultivating in the state close | similar to it. Therefore, as seen in the natural sediment environment, means for detecting the presence of bacteria and microorganisms in the presence of bacteria and other higher organisms (benthic organisms) and observing their activities Can not be used as.

  Therefore, the problem to be solved by the present invention is that the active state of various bacteria such as heterotrophic bacteria symbiotic with benthic organisms can be easily and accurately grasped without adversely affecting the bacteria. It is to provide a technique for observing bacterial activity.

The method for observing bacterial activity in the situation where the bacterium and benthic organism of the present invention coexist (hereinafter referred to as “bacterial activity observing method”) is a method in which a benthic organism can inhabit, An indicator that develops color, develops color, changes color, or emits light due to the physiological activity of bacteria is added to the transparent gel or the buffer coexisting with the transparent gel. With such a configuration, the indicator added to the transparent gel in which the benthic organisms can inhabit and the bacteria can grow at the same time or the buffer coexisting with the transparent gel is colored by the physiological activity of the bacteria. Since color development, color change, or light emission occurs, the state of color development, color change, color change, or light emission can be easily grasped with high accuracy by confirming the state of coloration, color change, color change, or light emission. In addition, it is possible to observe simply by adding an indicator to a transparent gel or a buffer solution coexisting with the transparent gel, and it is not necessary to modify the transparent gel or bacteria, so that the bacteria are not adversely affected. .

  Here, as the indicator, a compound that develops color, develops or emits light by oxidoreductase activity on bacterial cell membranes, a compound that develops by esterase activity in bacterial cells, or develops or emits light by bacterial extracellular hydrolase activity. A drug containing one or more of a compound, a compound colored by nucleic acid synthesis activity in bacterial cells, and a fluorescent substance having oxygen sensitivity can be used.

  When such a drug is used as an indicator, it exhibits oxidoreductase activity on bacterial cell membranes, esterase activity in bacterial cells, bacterial extracellular hydrolase activity, nucleic acid synthesis activity in bacterial cells, or oxygen consumption activity by bacterial cells. Depending on the degree, sensitive coloration, discoloration or luminescence occurs, so that the physiological activity of bacteria can be detected in a relatively short time.

  The compound that develops color, develops color or emits light by the oxidoreductase activity on the bacterial cell membrane includes any of tetrazolium compound, menadione and luminol, and the compound which develops color by the esterase activity in the bacterial cell is 6-carboxyfluorescein diacetate or A compound containing a lysorphine compound, which is colored or luminescent by bacterial extracellular hydrolase activity, contains a methylumbelliferyl compound or a lysorphine compound, and a compound colored by a nucleic acid synthesis activity in bacterial cells contains bromodeoxyuridine, Fluorescent materials having oxygen sensitivity include tris 1,7-diphenyl-1,10 phenatroline ruthenium (II) chloride.

  Examples of such drugs include “INT; 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride” from Dojindo Laboratories, “Cell Counting Kit-F” or 4-Methylumbelliferyl-β-D-galactoside from Nacalai Tesque, Inc. is preferred.

  On the other hand, image data obtained by imaging a portion that is colored, discolored, or emitted light by the indicator in the transparent gel can be binarized by a computer and displayed on a display device as an image. With such a configuration, it is possible to acquire and analyze the state of the colored, colored, discolored, or light-emitting portion generated in the transparent gel as image data.

  In this case, a calibration curve can be created from the image data and the spectroscopic data of the indicator, and the bacterial activity can be quantified based on the calibration curve. With such a configuration, it becomes possible to more accurately analyze and quantify the activity of bacteria.

  Furthermore, fine bubble generating means for supplying oxygen in the air into the buffer solution can also be used. With such a configuration, since oxygen in the air is supplied into the buffer solution, observation can be performed without impairing the physiological activities of benthic organisms and aerobic bacteria in the transparent gel.

  In this case, as the fine bubble generating means, the fluid swirl chamber generates a swirl flow generated around the axial center in the fluid swirl chamber by introducing the buffer solution and air into a cylindrical or rotating fluid swirl chamber. A fine bubble generator having a function of supplying a buffer solution mixed with fine bubbles into the buffer solution by discharging from a discharge port provided in a part of the can be used.

  If such a microbubble generator is used, the microbubbles supplied from the fluid swirling chamber of the microbubble generator to the buffer solution coexisting with the transparent gel together with the buffer solution are transferred to the transparent gel located below the buffer solution. Since it has the property of settling toward the bottom, it becomes possible to supply oxygen in the air more efficiently to benthic organisms and bacteria existing in the transparent gel. Therefore, continuous observation can be performed in a state in which the decrease in physiological activity of benthic organisms and aerobic bacteria due to the decrease in oxygen concentration is kept to a minimum.

  By the method for observing bacterial activity of the present invention, the activity state of various bacteria such as heterotrophic aerobic bacteria that exist symbiotically or coexist with benthic organisms can be accurately detected without adversely affecting the bacteria. Can be easily grasped.

  Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a diagram schematically showing changes in a culture medium and a buffer solution in the method for observing bacterial activity according to an embodiment of the present invention, and FIG. 2 is a boundary portion between the culture medium and the buffer solution shown in FIG. FIG.

  In the present embodiment, as shown in FIG. 1A, agar is used as a transparent gel 10 that can be inhabited by benthic organisms and serves as a bacterial culture medium, and seawater is used as a buffer solution 11 that coexists with the transparent gel 10. Is used. First, the adjustment procedure of the transparent gel 10 will be described. 0.7 g of agar (Bacto agar, Difco) is added to 100 ml of seawater, and after high-pressure sterilization treatment (held at 121 ° C. for 15 minutes), the bottom of the transparent pipe 15 having a predetermined length is plugged with a silicone rubber plug. The solution was poured into a culture vessel 16 formed by closing at 12 and solidified. At this time, about 40 ml of seawater in which agar is dissolved is poured into one culture vessel 16. Then, about 50 ml of seawater containing bacteria (not shown) was overlaid as the buffer solution 11 on the transparent gel 10 formed by solidifying the agar, and then 15 lobsters 17 were added.

Thereafter, a drug which is a kind of tetrazolium compound is added as an indicator into the culture vessel 16. This drug is 2- (p-iodophenyl) -3- (p-nitrophenyl) -5-phenyltetrazolium chloride (INT), but is not limited to this. In this case, the following two methods were adopted as a method of adding the indicator INT.
(1) After confirming that the coral 17 has burrowed into the transparent gel 10 and has settled, the final concentration of INT in the buffer solution (seawater) 11 coexisting on the transparent gel 10 is 0.1 weight. INT aqueous solution was added so that it might become% concentration.
(2) After the high-pressure sterilization treatment of seawater in which agar is dissolved, when the temperature of the transparent gel 10 reaches about 45 ° C., INT is added so that the final concentration of INT becomes 0.1% by weight. did.

  Next, the situation after adding the indicator INT and a predetermined time has elapsed will be described. As shown in the above (1), when INT is added to the buffer solution (seawater) 11, first, INT is reduced by the dehydrogenase activity on the bacterial cell membrane in the buffer solution (seawater) 11, and the red color is reduced. It became INT Formazan, and the buffer solution (seawater) 11 itself was dyed red. The time required for the red color to be discriminated visually was within 30 minutes. The red portion at this time is in a state as shown by fine dots on FIG.

  In this case, as indicated by the fine dots on FIG. 2, INT formazan was also formed in the burrow 19 of the swordfish 17 by the activity of the bacteria present there, and it turned red. It took about 2 to 3 hours for red to be discriminated by visual observation. This is because the buffer solution (seawater) 11 in the burrow 19 is exchanged with the buffer solution (seawater) 11 containing INT on the transparent gel 10 by the movement and watering activity of the carpenter 17 in the burrow 19. This is because it takes some time. From then on, the red part of each place became clearer with time.

  On the other hand, as shown in (2) above, when INT is added, the buffer solution (seawater) 11 does not become red as in the case of (1) described above, and the burrow 19 of the lobster 17 is a red color derived from INT formazan. I was dyed. In addition, since the degree of red staining of the burrow 19 is not uniform, the activity of bacteria present in the burrow 19 was determined to be spatially non-uniform.

  In this way, as shown in FIG. 1 (a), by using the transparent gel 10 as a bacterial medium as well as a substrate that can be inhabited by the benthic beetle 17, the agitation of the bentonite 17 An environment in which the action is simulated can be created in the culture vessel 16. In addition, as shown in FIG. 1B, the activity of heterotrophic bacteria occurring in the culture vessel 16 can be observed by the red coloration of the indicator INT. That is, by the method for observing bacterial activity of the present embodiment, the activity of bacteria in an environment where the bean shell 17 is present can be visualized non-destructively and easily evaluated. Further, as will be described later, these red portions are recorded as image data and can be quantified by analysis.

  In the present embodiment, the transparent gel 10 contains inorganic salts and organic substances used by heterotrophic bacteria to be used for the test. However, a bacterial metabolic activity indicator INT may be added to the transparent gel 10 in advance. Moreover, the upper part of the transparent gel 10 is filled with seawater as a buffer solution 11 for preventing the transparent gel 10 from drying and maintaining an appropriate osmotic pressure.

  As described above, in the transparent gel 10 in the culture vessel 16, a site colored in red by the bacterial activity indicator INT can be recorded and quantitatively analyzed as an image. In this case, an image of a portion colored in red is captured using an existing scanner, digital camera, microscope, or other imaging device, and binarization processing is performed on the image and analyzed.

  In addition, a calibration curve is created from the image data of the color development site of the indicator INT and the spectroscopic data of the indicator INT itself. The spectroscopic data of the indicator INT itself is spectroscopically quantified by extracting a known amount of the colored portion of the indicator INT from the transparent gel 10. If a calibration curve is created from the result and the result of the image data and the color development (discoloration) portion of the indicator INT in the unknown sample is quantified from the image data, the activity of the bacteria can be measured quantitatively.

  The colorless indicator INT used in the present embodiment is reduced by a bacterial respiratory enzyme, thereby producing a water-insoluble red precipitate called INT-formazan, which makes it possible to easily visualize the physiological activity of the bacteria. it can. Therefore, when INT is added to the buffer solution (seawater) 11, as shown in FIG. As a result, it was found that bacteria having high respiratory activity accumulate on the nest hole 19 and the tubule 21.

  In addition, in this embodiment, if the surface of the transparent gel (agar) 10 is made with a minute hole or notch with a needle or a knife to make a slight unevenness, the movement of the carpenter 17 to the inside of the nest hole 19 is achieved. It turns out that it is promoted. In addition, the seawater near the surface of the transparent gel 10 (interface in contact with the buffer solution (seawater) 11) is disturbed, or physical shaking (rotation, vibration, etc.) is given to the transparent culture vessel 16 itself containing the transparent gel 10. As a result, the movement of the cypress 17 into the nest 19 was promoted. It seems that it is possible to avoid disturbance on the surface of the lobster 17 by diving inside the transparent gel 10.

  Moreover, as a bacterial culture medium using the transparent gel 10, in addition to the cylindrical transparent pipe 15, the mixture of seawater and agar prepared as described above is poured between two transparent glass plates or acrylic plates. The experiment was conducted using a plate-shaped bacterial culture medium formed by solidification. These flat bacterial cultures are easy to focus on a wide range even when photographing at a close distance, so that they can be directly placed on the stage of a stereomicroscope and observed and photographed at a higher magnification. At that time, when oblique illumination was applied to the plate-shaped bacterial culture medium on the stage from obliquely above, it was possible to observe the color development site and obtain an image in a clearer state.

  On the other hand, if a portion of the transparent gel 10 containing the nest hole 19 made by benthic organisms (Itogokai 17) is taken out from the culture vessel 16 (for example, if it is cut out using a sterilized scalpel or the like), a specific place is examined in more detail. It is also possible to observe. The cut out transparent gel 10 part is infiltrated into a fixing solution such as formalin, so that the biological activity is stopped, stored for a long period of time, and can be observed and analyzed when necessary. Furthermore, by melting the agar component in the transparent gel 10 and centrifuging it, it is also possible to collect the viscous material or bacterial cells on the wall surface constituting the nest hole 19 of the benthic organism (Coleoptera 17) as a precipitated solid residue. Is possible. Moreover, these can be used for microscopic observation and component analysis.

Furthermore, it is also possible to include a specific compound in the transparent gel (agar) 10 and evaluate the influence of the compound on benthic organisms and bacteria. For example, sodium sulfide Na ₂ S · 9H ₂ O was added to the transparent gel 10 to evaluate the activity of forming burrows and the respiration activity of surrounding bacteria. It was found that the activity of bacteria was promoted.

  Next, referring to FIG. 3 to FIG. 7, a method for observing bacterial activity when a microbubble generator for supplying oxygen in the air into a buffer solution, which is another embodiment of the present invention, is used. Will be described. 3 is a schematic diagram showing a method for observing bacterial activity when a microbubble generator is used, FIG. 4 is a perspective view of the microbubble generator shown in FIG. 3, and FIG. 5 is a cross-sectional view taken along line AA in FIG. 6 is a cross-sectional view taken along the line BB in FIG. 5, and FIG. 7 is a diagram schematically showing a state of generation of fine bubbles in the fine bubble generator shown in FIG.

  As shown in FIG. 3, the buffer solution 11 is stored in the storage tank 22, and a plurality of culture vessels 23 containing the transparent gel 10 fill the interior with the buffer solution 11 and the entire culture vessel 23 is completely buffered. It is disposed in the storage tank 22 so as to be immersed in the liquid 11. In addition, the fine bubble generator 13 is immersed in the buffer solution 11 in the storage tank 22, and a suction pipe 24 for sucking up the buffer solution 11 with the electric pump P is provided. A filter 24f is attached to the. A liquid supply pipe 18 is provided between the pump P and the fine bubble generator 13, and an air supply pipe 14 for supplying air to the fine bubble generator 13 is provided from the fine bubble generator 13 to the outside of the storage tank 22. A filter 14f is attached to the tip of the filter.

  In such a state, when the pump P is operated, the buffer solution 11 in the storage tank 22 is sucked into the suction pipe 24 through the filter 24f, and is finely immersed in the buffer solution 11 through the liquid supply pipe 18. It is supplied to the bubble generator 13. At this time, due to the negative pressure generated in the fine bubble generator 13, air in the atmosphere is sucked into the air supply pipe 14 through the filter 14 f and enters the fine bubble generator 13 in the buffer solution 11 through the air supply pipe 14. Supplied. Thereby, as shown in FIGS. 3 and 4, the buffer solution 11 mixed with the fine bubbles NB is discharged from the discharge port 28 of the fine bubble generator 13 into the buffer solution 11 in the storage tank 22. A mechanism for generating the fine bubbles NB by the fine bubble generator 13 and a mechanism for generating a negative pressure in the fine bubble generator 13 will be described later.

  As shown in FIGS. 5 and 6, the fine bubble generator 13 is provided at a cylindrical peripheral wall 25d provided so as to surround the virtual center line 25c, and at both ends of the peripheral wall 25d in the direction of the virtual center line 25c. The fluid swirl chamber 25 formed by the partition walls 25a and 25b communicates with the fluid swirl chamber 25 to introduce a buffer solution into the fluid swirl chamber 25 along a direction that is twisted with respect to the virtual center line 25c. Provided in the fluid supply chamber 18, the air supply tube 14 connected to one partition wall 25 a of the fluid swirl chamber 25 for introducing air into the fluid swirl chamber 25, and the other partition wall 25 b of the fluid swirl chamber 25. And a discharge port 28. The liquid supply pipe 18 communicates with the fluid swirl chamber 25 via the liquid inlet 27, and the air supply pipe 14 communicates with the fluid swirl chamber 25 via the gas inlet 26.

  As shown in FIG. 3, when the pump P is operated in a state where the fine bubble generator 13 and the filter 24 f of the suction pipe 24 are immersed in the buffer solution 11 in the storage tank 22, the filter 24 f and the filter 24 f from the storage tank 22 are operated. The buffer solution 11 sucked through the suction pipe 24 flows into the fluid swirl chamber 25 from the liquid inlet 27 through the liquid supply pipe 18, and as shown in FIGS. A swirling flow R is generated in the 25. Then, a substantially cylindrical negative pressure cavity V appears along substantially the virtual center line 25c of the swirling flow R. One end of the negative pressure cavity V is located near the gas inlet 26 established in the partition wall 25 a of the fluid swirl chamber 25, and the other end of the negative pressure cavity V is the fluid swirl chamber 25. The end of the negative pressure cavity V located near the discharge port 28 provided in the partition wall 25b and in the vicinity of the discharge port 28 is constricted.

  Since the negative pressure of the negative pressure cavity V appearing in the fluid swirl chamber 25 as described above causes a negative pressure also in the vicinity of the gas inlet 26, the suction force caused by this negative pressure causes the air supply pipe 14 to pass through. The air sucked from the atmosphere continuously flows from the gas inlet 26 into the negative pressure cavity V in the fluid swirl chamber 25, and swirls R together with the buffer solution 11 introduced into the fluid swirl chamber 25. Form.

  The air that has flowed into the negative pressure cavity V is entrained in the swirl flow R generated in the fluid swirl chamber 25 and discharged from the discharge port 28. At this time, the end of the negative pressure cavity V on the discharge port 28 side is threaded by the swirling flow R to become fine bubbles NB, and together with the buffer solution 11 forming the swirling flow R, becomes the buffer solution 11 mixed with the fine bubbles NB. Then, it is discharged from the discharge port 28 into the buffer solution 11 in the storage tank 22.

  In this manner, oxygen can be supplied and dissolved in the buffer solution 11 in the storage tank 22 by discharging the fluid (buffer solution 11) mixed with the fine bubbles NB into the buffer solution 11 from the discharge port 28. For this reason, the buffer solution 11 mixed with the fine bubbles NB having a high dissolved oxygen concentration circulates uniformly in the storage tank 22, and the transparent gel 10 in the plurality of culture vessels 23 immersed in the buffer solution 11. Can supply oxygen evenly. Therefore, as shown in FIG. 3, when an experiment is performed using a plurality of culture vessels 23, it is possible to equalize the experimental conditions for each culture vessel 23, which is effective in improving the experiment accuracy. In addition, since the plurality of culture vessels 23 are immersed in the buffer solution 11 in the storage tank 22 and the fine bubble generator 13 is inserted into the buffer solution 11 and the pump P is operated, it can be used very much. Simple.

  Further, since the fine bubble generator 13 has a simple structure in which the liquid introduction port 27, the gas introduction port 26, and the discharge port 28 are opened in the substantially cylindrical fluid swirl chamber 25, the handling is easy and the buffer solution 11 and there is no fine channel that is likely to be clogged with foreign matter that flows in with air, so that periodic maintenance is also unnecessary.

  Furthermore, as shown in FIG. 6, the gas introduction port 26 opened in the partition wall 25 a of the fluid swirl chamber 25 is arranged to project inward along the virtual center line 25 c of the fluid swirl chamber 25, and the fluid swirl chamber A smoothly curved concave surface 29 is provided between the peripheral wall 25d of 25 and the gas inlet 26. For this reason, as shown in FIG. 7, air is introduced from the end of the negative pressure cavity V formed in the fluid swirl chamber 25 on the side of the partition wall 25a, and finer in the extending direction of the end on the side of the partition wall 25b. The buffer solution 11 mixed with the bubbles NB is discharged. Therefore, the negative pressure cavity V continues to exist stably in the vicinity of the imaginary center line 25c of the fluid swirl chamber 25, and both ends thereof are also stably positioned in the vicinity of the discharge port 28 and the gas inlet 26.

  As described above, the gas introduction port 26 is arranged so as to protrude to the inside of the fluid swirl chamber 25 and the concave curved surface 29 is provided, so that the end of the negative pressure cavity V on the gas introduction port 26 side irregularly moves. There is nothing to do. For this reason, cavitation erosion or the like does not occur in the partition walls 25a and 25b of the fluid swirl chamber 25, and excellent durability is obtained. As shown in FIGS. 5 and 6, a preliminary swirling portion 25 p having a larger inner diameter than other regions is provided in a region near the partition wall 25 b of the fluid swirl chamber 25. For this reason, the buffer solution 11 introduced from the liquid inlet 27 can be once rectified in the preliminary swirl unit 25p and then introduced into the entire fluid swirl chamber 25. As a result, the pressure fluctuation of the buffer solution 11 introduced from the liquid inlet 27 is buffered, and the movement of the negative pressure cavity V due to this pressure fluctuation can be prevented, so that cavitation erosion does not occur.

  As shown in FIG. 6, in the fine bubble generator 13, the opening area of the liquid introduction port 27 is larger than the opening area of the discharge port 28, and thus the buffer solution 11 fed into the fluid swirl chamber 25. The outlet of the buffer solution 11 mixed with fine bubbles NB is smaller than the inlet of. Therefore, the fluid pressure in the fluid swirl chamber 25 is increased by the pressure of the buffer solution 11 fed by the pump P, and the flow of the buffer solution 11 mixed with the fine bubbles NB discharged from the discharge port 28 is increased. The circulation of the buffer solution 11 in the tank 22 becomes good, which is effective for making the experimental conditions uniform.

  Next, with reference to FIGS. 8 to 11, other fine bubble generating means having the same function as the fine bubble generator 13 described above will be described. FIG. 8 is a side view showing a fine bubble generator according to another embodiment, FIG. 9 is a partially omitted plan view of the fine bubble generator shown in FIG. 8, and FIG. 10 constitutes the fine bubble generator shown in FIG. FIG. 11 is a sectional view taken along line CC in FIG. 10.

  As shown in FIGS. 8 and 9, the fine bubble generating device 1 includes a liquid-proof liquid pump 2, a liquid-proof electric motor 3 that is a drive for operating the liquid pump 2, and a fine-bubble generator 4. , Are integrally connected. The electric motor 3 is supplied with power from a generator (not shown) arranged outside via the power cord 5 and can be turned on and off by a switch (not shown). The liquid pump 2 is coaxially connected to the rotating shaft (not shown) of the electric motor 3, and the buffer solution 11 such as seawater sucked from the suction port 2 a is supplied to the liquid of the fine bubble generator 4 via the discharge part 8. Supply to the introduction path 35.

  The fine bubble generator 4 has a substantially cylindrical shape and is connected to the tip of an air supply pipe 9 for introducing air in the atmosphere fed from a gas pump (not shown) arranged outside. The base end portion of the air supply pipe 9 is connected to a gas pump. A check valve 33 is provided in the middle of the air supply pipe 9 to prevent the buffer solution 11 from flowing backward.

  As shown in FIGS. 10 and 11, the fine bubble generator 4 includes a cylindrical casing 4 a containing a fluid swirl chamber 34 in which a fluid (buffer solution and air) can swirl around an axis S, and a fluid swirl chamber 34. In order to introduce the buffer solution 11 into the fluid swirl flow R to generate the fluid swirl flow R, a liquid introduction path 35 for ejecting the buffer solution 11 into the fluid swirl chamber 34 and a fluid swirl chamber 34 to introduce air into the fluid swirl chamber 34 An air introduction passage 36 formed in communication with the discharge passage 37 and a discharge passage 37 formed at the end of the fluid swirl chamber 34 in the axial center S direction. The discharge path 37 is formed at the center part (intersection part with the axis S) of the planar partition wall 37a orthogonal to the axis S of the cylindrical casing 4a.

  The liquid introduction path 35 has a cylindrical shape whose outer diameter is smaller than that of the cylindrical casing 4 a, and a connection part 35 c to the discharge part 8 of the liquid pump 2 is provided at the base end part, and the tip part thereof is the fluid swirl chamber 34. Is disposed so as to protrude into the fluid swirl chamber 34 at the end opposite to the discharge path 37, and its tip is closed by a closing plate 35 a. As shown in FIGS. 10 and 11, the buffer solution 11 is ejected to the outer periphery of the liquid introduction path 35 located in the fluid swirl chamber 34 along a direction that forms a twist position with the axis S of the fluid swirl chamber 34. A plurality of jet outlets 35b are provided.

  As shown in FIG. 11, the air introduction path 36 penetrates through the side surface portion of the liquid introduction path 35 and enters the inside thereof, and bends at right angles to the axis S direction. Is opened on the surface side (the fluid swirl chamber 34 side). A base end portion of the air supply pipe 9 is detachably connected to an air introduction path 36 protruding from a side surface of the liquid introduction path 35. Therefore, the air sent from the air supply pipe 9 is directly supplied into the fluid swirl chamber 34 through the air introduction path 36 from the tip opening 36a opened on the surface of the closing plate 35a.

  As shown in FIG. 11, outside the discharge path 37 of the cylindrical casing 4a containing the fluid swirl chamber 34, a disk-like shape having a plane 38a intersecting with the axis S at a position facing the discharge path 37. A guide member 38 is disposed. The guide member 38 is joined to the tip end portion of the cylindrical casing 4a via three arc-shaped connecting members 38b and 38c, and from three outlets 39 formed between the connecting members 38b and 38c. The buffer solution 11 mixed with fine bubbles NB, which will be described later, can be blown out. Note that these three outlets 39 are arranged at intervals of 90 degrees in three directions excluding the arrangement direction of the electric motor 3 in a plan view.

  As shown in FIG. 3, when the fine bubble generating device 1 is put into the buffer solution 11 in the storage tank 22 (see FIG. 3), held in the standing state in the buffer solution 11, and the electric motor 3 is operated, The buffer solution 11 sucked from the suction port 2a of the pump 2 flows from the discharge part 8 into the liquid introduction path 35, and is ejected into the fluid swirl chamber 34 through the ejection port 35b. At this time, since the ejection direction of the buffer solution 11 is a direction that forms a twisted position with the axis S of the fluid swirl chamber 34, a liquid flow swirling around the axis S is generated in the fluid swirl chamber 34. At the same time, a part of the liquid flow is discharged from the discharge path 37 into the buffer solution 11.

  At this time, as shown in FIG. 11, a negative pressure cavity V is generated near the axis S in the fluid swirl chamber 34, and the presence of this negative pressure cavity V creates a negative pressure in the fluid swirl chamber 34. Therefore, air in the atmosphere is smoothly introduced via the air introduction path 36 and the air supply pipe 9 that communicate with the fluid swirl chamber 34, and flows into the fluid swirl chamber 34 from the tip opening 36 a of the air introduction path 36. Thereby, in the fluid swirl chamber 34, a swirl flow (for example, a swirl two-layer flow) composed of a negative pressure cavity V positioned in the vicinity of the axis S and an air-mixed buffer solution that rotates around the negative pressure cavity V. ) Is formed.

  In a state where such a swirl flow is formed in the fluid swirl chamber 34, the air introduced into the fluid swirl chamber 34 via the tip opening 36 a of the air introduction path 36 is the shearing action of the swirl flow described above. And the fluid swirl flow R is swirled at high speed in the fluid swirl chamber 34. The fluid swirl flow R eventually moves toward the partition wall 37 a of the fluid swirl chamber 34, converges toward the discharge path 37 by contacting the partition wall 37 a, and passes through the discharge path 37 narrower than the inner diameter of the fluid swirl chamber 34. As a result, the buffer solution is mixed with fine bubbles NB rotating at a higher speed, and then discharged. Thus, in order to release the fluid (buffer solution 11) mixed with the fine bubbles NB from the discharge path 37 into the buffer solution 11, as in the case of the fine bubble generator 13 shown in FIG. Oxygen can be supplied and dissolved.

  Next, with reference to FIG. 12 to FIG. 15, other fine bubble generating means having the same function as the fine bubble generator 13 described above will be described. 12 is a front view showing a fine bubble generator according to another embodiment, FIG. 13 is a perspective view of the fine bubble generator shown in FIG. 12, FIG. 14 is a sectional view taken along the line DD in FIG. 13, and FIG. FIG. 13 is a schematic diagram showing an operating state of the fine bubble generator shown in FIG.

A microbubble generator 40 shown in FIGS. 12 to 15 is provided with a cylindrical fluid swirl chamber 42 in which a fluid (buffer solution and air) can swirl within a substantially rectangular parallelepiped casing 41, and the axis of the fluid swirl chamber 42. An air introduction path 44 for introducing air is opened in the normal direction on the inner peripheral surface 42a of the central portion in the center S direction, and a fluid swirl chamber is located at both ends of the air introduction path 44. In the tangential direction of the inner peripheral surface 42a of 42, two liquid introduction paths 43 capable of introducing a buffer solution are established.

  The liquid introduction path 43 and the air introduction path 44 are formed through the casing 41, and a liquid introduction pipe 43a and an air introduction pipe 44a are connected to respective opening portions of the outer surface of the casing 41. The two liquid introduction pipes 43a are connected to the liquid delivery pipe 43b in a state of being unified on the upstream side, and the air introduction pipe 44a is extended in the direction of the liquid delivery pipe 43b as it is.

  Further, at both ends of the fluid swirl chamber 42 in the direction of the axis S, planar partition walls 41a orthogonal to the axis S are provided, and in the central part of these partition walls 41a (intersection with the axis S). The circular discharge paths 45 are opened, and the guide pipes 46 are connected to the two discharge paths 45, respectively. As will be described later, the guide tube 46 is communicated so as to protrude linearly along the discharge direction of the buffer solution mixed with the fine bubbles NB discharged from the discharge path 45, and the discharge direction is changed by the guide tube 46. It is regulated.

  Similar to FIG. 3, the fine bubble generator 40 is immersed in the buffer solution 11 in the storage tank 22, and buffered from a liquid pump (not shown) arranged outside via the liquid feed pipe 43 b and the liquid introduction pipe 43 a. When the liquid 11 is pumped and seawater is pumped from the liquid introduction path 43 into the fluid swirl chamber 42 and air is introduced into the fluid swirl chamber 42 via the air introduction pipe 44a, the fluid swirl A fluid swirl flow R around the axis S is generated in the chamber 42, and a negative pressure cavity V is formed in the vicinity of the axis S.

  The air flowing into the fluid swirl chamber 42 from the air introduction path 44 is finely crushed by the shearing action of the fluid swirl flow R, and rotates around the negative pressure cavity V to become fine bubbles NB. It is discharged from the path 45 as seawater mixed with fine bubbles NB. Seawater mixed with fine bubbles NB discharged from the discharge path 45 is discharged into the buffer solution 11 in the storage tank 22 while being guided by the guide pipe 46. As a result, the amount of dissolved oxygen in the buffer solution 11 is increased, and the same effect as when the fine bubble generator 13 is used can be obtained.

  The present invention can be widely used as a means for observing the active state of heterotrophic bacteria that inhabit benthic organisms in coastal sediments.

It is a figure which shows typically the change of the culture medium and the buffer solution in the observation method of the bacterial activity which is embodiment of this invention. It is a partially expanded sectional view which shows the boundary part of the culture medium shown in FIG.1 (b), and a buffer solution. It is a schematic block diagram which shows the observation method of the bacterial activity using a microbubble generator. It is a perspective view of the fine bubble generator shown in FIG. It is the sectional view on the AA line in FIG. It is the BB sectional view taken on the line in FIG. It is a figure which shows typically the fine bubble generation state in the fine bubble generator shown in FIG. It is a side view which shows the fine bubble generator which is other embodiment. FIG. 9 is a partially omitted plan view of the fine bubble generating device shown in FIG. 8. It is a partially cutaway side view of the fine bubble generator which comprises the fine bubble generator shown in FIG. It is CC sectional view taken on the line in FIG. It is a front view which shows the fine bubble generator which is other embodiment. It is a perspective view of the fine bubble generator shown in FIG. It is the DD sectional view taken on the line in FIG. It is a schematic diagram which shows the operating state of the fine bubble generator shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fine bubble generator 2 Liquid pump 2a Suction port 3 Electric motor 4a Cylindrical casing 5 Power cord 8 Liquid discharge part 9 Air supply pipe 10 Transparent gel 11 Buffer liquid 12 Silicon rubber stopper 4, 13, 40 Fine bubble generator 14 Air supply pipe 14f, 24f Filter 15 Transparent pipe 16, 23 Culture vessel 17 Lepidoptera 18 Liquid supply pipe 19 Nest hole 20 Feces 21 Fistula 22 Reservoir 24 Suction pipe 25, 34, 42 Fluid swirl chamber 25a, 25b, 37a, 41a Partition 25c Virtual center Line 25d Peripheral wall 25p Preliminary swirl chamber 26 Gas introduction port 27 Liquid introduction port 28 Discharge port 29 Concave surface 33 Check valve 35, 43 Liquid introduction route 35a Blocking plate 35b Jet port 35c Connecting portion 36, 44 Air introduction route 36a Front end opening portion 37, 45 Discharge path 38 Guide member 38a Plane 38b, 38c Connecting member 39 Outlet 41 Casing 42a Inner peripheral surface 43a Liquid introduction pipe 44a Air introduction pipe 43b Liquid feed pipe 46 Guide pipe NB Fine bubble P Pump R Swirling flow S Shaft center V Negative pressure cavity

Claims (6)

Bacteria characterized by adding an indicator that develops color, develops color, changes color, or emits light according to the physiological activity of bacteria in a transparent gel that can be inhabited by benthic organisms and that is used as a bacterial culture medium or in a buffer that coexists with the transparent gel Method for observing bacterial activity in the presence of terrestrial and benthic organisms . As the indicator,
A compound that develops color, develops color or emits light by oxidoreductase activity on the bacterial cell membrane,
Compounds colored by esterase activity in bacterial cells,
A compound that is colored or luminescent by bacterial extracellular hydrolase activity,
Compounds colored by nucleic acid synthesis activity in bacterial cells,
A fluorescent substance having oxygen sensitivity,
A method for observing bacterial activity in a situation in which bacteria and benthic organisms coexist according to claim 1, wherein a drug containing at least one of the above is used. 3. The image data obtained by imaging a portion colored, colored, discolored, or emitted light by the indicator in the transparent gel is binarized by a computer and displayed as an image on a display device. Method for observing bacterial activity in a situation where both bacteria and benthic organisms coexist .   4. The method for observing bacterial activity according to claim 3, wherein a calibration curve is created from the image data and spectroscopic data of the indicator, and the bacterial activity is quantified based on the calibration curve. Bacterial activity in a situation in which bacteria and benthic organisms coexist in any one of claims 1 to 4, characterized by using fine bubble generating means for supplying oxygen in the air into the buffer solution. Observation method. As the fine bubble generating means, a swirl flow generated around the axial center in the fluid swirl chamber by introducing the buffer solution and air into a cylindrical or rotating fluid swirl chamber is part of the fluid swirl chamber. 6. The bacterium and benthic organism according to claim 5, wherein a microbubble generator having a function of supplying a buffer solution containing a mixture of microbubbles into the buffer solution by being discharged from a discharge port established in the above is used. A method for observing bacterial activity under coexisting conditions .

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