JP2000228976A - Isothermal culture for measuring bod and apparatus therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out isothermal culture while preventing the evaporation of water from the stopper of a BOD culture bottle having no collar and preventing the intrusion of oxygen in air into the inside of the culturing bottle in measuring BOD in an isothermal tank. SOLUTION: Culture bottles 1 having no collar for BOD measurement are placed in a light-shaded heat-insulated air tank 6. By spraying constant- temperature water continuously or intermittently using a water-temperature controlling water circulating apparatus 8, the temperatures of the inside of the tank and the culture bottles 1 are kept constant, and at the same time, water sealing water 13 is formed by making the upper part of the contact between the mouth of the culturing bottle 1 and the stopper wet. Thus, isothermal culture is carried out while preventing the evaporation of water from the periphery of the stopper of the culture bottle 1 and preventing the intrusion of oxygen in air into the inside of the culture bottle 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture apparatus and method for measuring BOD (biochemical oxygen demand).

[0002]

2. Description of the Related Art B, one of the indicators of environmental water pollution,
The OD is measured by placing the water to be measured in a glass culture bottle with a stopper, culturing bacteria contained in the water at a constant temperature under light shielding, and measuring the amount of oxygen consumed by the contained organic matter. During this culture, water at the contact surface between the bottle mouth and the stopper evaporates, and oxygen in the air penetrates into the inside of the bottle through the resulting gap, causing an error in the measured value. In order to prevent this, the following two methods are used for the culture method.

[0003] b. The sample to be measured is placed in the culture bottle (1) without the collar shown in Fig. 3, plugged, immersed in a thermostatic water bath so that the mouth of the bottle is below the surface of the water, and the stopper and the bottle are contacted with the thermostatic water. A method of culturing while preventing the evaporation of water on the contact surface by sealing the part with water and preventing the intrusion of oxygen into the culture bottle.

[0004] b. As shown in FIG. 3, a colored culture bottle (4) with a glass collar (2) integral with the bottle so as to surround a stopper around the mouth of the culture bottle, or
A measurement sample is put into a culture bottle (4) with a collar attached with a detachable collar (3) made of rubber or a soft synthetic resin around a stopper of the culture bottle (1) without a collar. ) To seal the water between the bottle's mouth and the stopper, preventing water evaporation and preventing oxygen in the air from entering the inside of the culture bottle. Or a method of culturing in a thermostatic chamber.

[0005]

The prior art has the following problems when measuring a large number of culture bottles.

The above a. When measuring a large number of samples, dozens of them are placed in a case and immersed in a constant temperature water bath for handling and efficiency.Therefore, when taking out the case from the water, a sudden load is applied on the water surface. , Burden on the body. Furthermore, it is necessary and troublesome to keep the water on the tank for a few seconds in order to drain the water from the case. In addition, the floor surface is wet with dripping water, which makes the work difficult. Further, since constant temperature water is soiled with time, it is necessary to periodically exchange a large amount of water. Further, when culturing a large number of culture bottles, a large installation area is required because the water tank cannot be deeply concealed from the point of putting the culture bottles in and out.

Further, b. In, there is a troublesome work of adding water to the collar portion before culturing and discarding the water after culturing. Further, when the color portion is made of glass, it is easily broken.

The present invention has been made in view of the above-mentioned problems of the prior art, and prevents the penetration of oxygen in the air into the bottle by preventing evaporation of water around the stopper of the culture bottle. When performing constant temperature cultivation, it is possible to eliminate the hassle of water sealing work for each bottle, easily perform water sealing, and easily and quickly dozens of pieces into and out of the heat insulation tank at a time. It is an object.

[0009]

Means for Solving the Problems In order to achieve the above object, the present invention comprises the following means.

A colorless culture bottle (hereinafter referred to as a culture bottle) in which a sample is filled in a sealed light-shielded warm air tank.
During the culture, constant temperature water is sprayed continuously or intermittently in the tank, and a water-wet state is constantly formed on the upper part of the contact between the mouth and the stopper of the bottle. Further, the spray keeps the inside of the tank in a state of saturated steam, and prevents evaporation of water attached to the upper part of the contact between the mouth and the stopper of the bottle. Together, the water seal state of the contact peripheral portion is maintained to prevent the generation of a gap due to the evaporation of water, thereby blocking the invasion of oxygen in the air into the culture bottle.

The spray of the constant-temperature water set at a predetermined temperature raises the temperature of the air in the tank to a predetermined temperature, and the stirring of the air in the tank by the spray water keeps the temperature in the tank uniform and constant. or,
The constant temperature water is poured into the bottle to help maintain the culture bottle at a predetermined temperature. In this manner, constant temperature culture for BOD measurement is performed.

After the incubation at constant temperature for a certain period of time, when the cap is opened for measurement of oxygen in water in the culture bottle, the amount of water sealed on the upper part of the contact between the mouth and the cap of the bottle is very small and enters into the bottle. There is nothing.

The state of spraying is from a mist to a shower. The fine mist goes behind the culture bottle. Any of the sprays in the form of a shower can be used because water droplets collide with the culture bottle and bounce off the culture bottle, and any of them can be used. However, a shower is preferable to quickly reach the set temperature of the culture bottle. However, since the amount of water in the shower is larger than that in the fog, the capacity of the water circulation pump is increased. In the case of a large-sized culture device, it is desirable to mix the mist and the shower mist. Further, the mist state and the shower state may be switched and used.

In order to set the position, number, direction, spraying power, spraying angle and droplet size of the spray ports, the following matters must be taken into consideration, as the water spray falls on the top of all the culture bottles. No. The installation position of the spray port is the upper surface and the side surface in the tank. The selection of the surface and the number and position of the spray ports are determined by the size and shape of the tank, the angle of spraying, the particles and the strength, and the like. Furthermore, it depends on the installation status of the culture bottle.
Since there are so many factors, a trial and error is required for the setting. In order not to hinder the spraying, it is preferable to place the culture bottle at a position about 10 cm or more from the spraying port.

In the case of a small tank of about 20 to 30 culture bottles, two or three spray ports may be provided on one of the upper surface or side surface. However, if the tank is large, a number of spray ports are provided on the side and top. In addition, it is also possible to install a spray port at a place other than the side where the culture bottle can be taken in and out. The installation interval of the spray ports depends on the capacity of the spray ports and the size and shape of the tank, but is installed at intervals of about 10 to 60 cm. In the case of the ceiling of the tank, the spray port is installed downward, but the one near the side wall is inclined inward. When the spray port is provided on the side wall, the direction is directed toward the culture bottle, but it is preferable that the upper one is downward, the middle one is almost horizontal, and the lower one is upward.

As the spray port, a normal spray nozzle for disinfection and painting, a rotary watering nozzle, a shower, or the like is used.
A spraying distance of about 30 to 200 cm and a spread angle of about 30 to 130 degrees are used.

The water spray keeps the humidity in the tank at a saturation level, thereby suppressing evaporation of water from the culture bottle. Therefore, if the inside of the culture bottle reaches a constant temperature, intermittent spraying may be used. The intermittent interval depends on the heat retention structure of the heat retention tank and the amount of water sealing water. When the lid or door of the heat retaining tank is opened, the saturated state of water vapor and the set temperature are broken, so it is necessary to spray immediately. Usually, the control of the intermittent spraying is performed by a temperature sensor in the tank with emphasis on the temperature. ,

If the spraying is stopped several tens of minutes before the culture bottle is taken out and kept at a constant temperature and a water vapor saturated state, it is possible to prevent dripping from the culture bottle outside the tank when the culture bottle is taken out.

The wall structure for maintaining the temperature of the heat retaining air tank depends on the spray amount of room temperature and constant temperature water. That is, when the room temperature at which the bath is installed is close to the set temperature in the bath, a special heat retaining structure need not be provided. If the difference between room temperature and set temperature is large,
Either strengthen the heat retaining structure, increase the amount of constant temperature water spray, or allow both to achieve the set constant temperature.

The constant-temperature water used for spraying passes through a floor provided at the lower part of the tank so that the constant-temperature water does not stay, passes through a water storage tank provided at the bottom of the tank or outside the tank, and is subjected to water temperature control and water circulation. It is recycled in the equipment. The following floor structures are used. When a large tank is used with a trolley, the floor structure must be strong enough to withstand the weight of the trolley and have a surface structure that does not hinder the passage of wheels. The plate-shaped perforated plate provided with holes is provided with a plurality of holes in consideration of the size and the number of holes having a capacity for passing circulating water. Also, a thin groove for flowing water may be provided on the upper surface of the board to guide the circulating water to the periphery of the board, and may be guided to a water storage tank from a peripheral hole. Alternatively, the floor may be slightly tilted so as not to hinder the fixation and movement of the bogie, and the water may be led to the end of the floor by the tilt, and the water may be led to the water storage part from the hole provided therein. It is preferable that the slope be inclined backward from the viewpoint of use of the bogie. The slit used is a scaly one.

In a small-sized culture apparatus, the culture bottle is put in or out of a cage as it is or in a basket, through an opening provided in a tank. The baskets can be stacked. In the case of a large culture tank, a door for a culture bottle such as a sliding door is provided on the side of the tank. A plurality of water-permeable cases accommodating a large number of culture bottles are placed on a plurality of carts having multi-stage shelves, and the carts are put in and out of the tank. Also, several shelves may be provided inside the tank, and a large number of the cases may be placed there. These shelves have a structure in which water does not stay like the above-mentioned floor structure. However, when the shelf area is small and water stays negligible, these considerations need not be considered.

As the constant temperature water, the water adjusted by a water temperature controller outside the tank is circulated and used, but a water temperature controller may be installed inside the tank. The water circulation pump may be installed inside or outside the tank. It is preferable to use a device in which the water temperature control and the water circulation pump are integrated.

The location of the reservoir for the constant temperature water may be inside or outside the tank. In the case of the inside, the heat retaining structure is unnecessary, but since the floor surface in the tank is higher than the outside of the tank, an inclined plate for raising and lowering is required outside the tank when using the bogie. If the water storage tank inside or outside the tank is installed below the external floor surface, the floor for elevating is not required.

If the tank is a heat-retaining air tank with an air temperature controller, it is not necessary to specially install the above-mentioned water temperature controller. In this case, if the water in the water storage section installed at the bottom of the tank or outside the tank is circulated, the circulating water reaches a predetermined temperature by the constant temperature air.
Therefore, if the water is sprayed and circulated before using the apparatus, the circulating water becomes constant temperature water by the constant temperature air. From that point, a culture bottle is put in and the culture is started.

For setting the temperature of the air, the air may be circulated to a thermostat installed outside the tank, or a heater and a cooler may be installed in the tank, and these may be controlled by sensors installed in the tank. A fan for air stirring is installed in the tank, but if it is not a large tank, there is air stirring by water spray,
It is not always necessary to install a fan.

In a normal culture bottle, water sealing can be performed at the mouth of the bottle, but in some cases, the structure of the contact upper portion between the mouth and the stopper is difficult to form water sealing. In such a case, the problem can be solved by using a structure in which the contact upper portion has the following structure and has a structure in which water sealing water is actively formed.

If the periphery of the shoulder of the stopper is cut off in a triangular shape with a sectional area, and a valley structure is formed in which the upper part of the inner surface of the mouth of the culture bottle is not in contact with the stopper, a puddle state is formed in that part. The seal becomes more complete. However, this valley structure must be as shallow as possible so as not to impair the function of the plug.

If the upper end of the stopper in contact with the mouth of the bottle is lowered below the upper end of the inner surface of the bottle, that is, if the stopper is inserted deeply, a puddle is formed on the upper part of the stopper.
Can be sealed with water. If the structure is pushed deeply into the darkness, the amount of water sealed will increase, and it will be troublesome to remove the plug when opening the plug. Further, the function of the plug will be impaired, so it is important to make it as shallow as possible.

In the above case, removal of the puddle at the time of opening the plug is as follows.
Since it is a small amount, it can be easily removed by tilting, but can be easily blown off by air spray or air jet.

[0030]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described with reference to the drawings based on embodiments. FIG. 1 is made of a foamed resin having a thickness of 30 mm and has inner dimensions of 250 × 360 × 3.
This is a culture tank using a small heat-retaining air tank 6 of 00 mm.
It has a lid 7 for taking the culture bottle 1 in and out. FIG. 2 shows a planar structure with the lid 7 removed. Six culture bottles 1 are housed, and constant temperature water at 20 ° C. ± 1 ° C. is sent under pressure in the direction of the arrow through the circulation pipe 9 by the water temperature control / water circulation device 8 installed outside,
Thermostatic water is sprayed into the tank from two water spray ports 10 installed in a circulation pipe 9 installed in the upper part of the tank. The spray port is the spray port of a conventional shoulder-type pesticide sprayer. Spray angle about 110
Spray continuously at a speed of about 500 mm.
At room temperature of 25 ° C., the temperature in the bath reached 20 ° C. ± 1 ° C. after 3 minutes. The sprayed constant temperature water reaches the upper contact portion 11 between the mouth and the stopper of the culture bottle 1, and as shown in an enlarged view 12, creates a water seal water 13 at the upper contact portion 11 between the bottle and the stopper.
A water seal is performed to prevent oxygen in the air from entering the culture bottle 1. The water sealing water 13 usually has a raised shape due to the surface tension of the water. The sprayed constant temperature water comes into contact with the culture bottle 1 and, together with the constant temperature air, adjusts the liquid temperature in the culture bottle 1 to a set temperature of 20 ° C.
Bring to ± 1 ° C. The constant temperature water used was a hole 35 (diameter 15 m) of the perforated plate 14 installed 5 cm above the bottom of the tank.
m are provided evenly), collected in the lower water storage section 15 and passed through the circulation pipe 9 to control the water temperature.
The water is recirculated in the water circulation device 8. The water level of the water storage section 15 was set to about 3 cm, and the water temperature control / water circulation device 8 was set so that the water circulation pump did not suck air.

6 g of sterile distilled water containing 2.4 mg / l of dissolved oxygen
The dissolved oxygen after being placed in the culture bottle 1 and cultured for 5 days under the above conditions was 2.3, 2.4, and 2.4 mg / l, respectively, and there was no oxygen inflow and out during the culture period.

FIG. 4 shows the contact upper part 11 between the mouth and the stopper of the culture bottle.
Fig. 1 shows an example of a structure in which water sealing water is actively made. As shown in an enlarged view 16 of the upper part 11 of contact between the mouth of the bottle and the stopper, the periphery of the shoulder of the stopper is cut out in the shape of a triangular cross section 19. By doing so, the water seal water 18a is formed between the upper end portion of the inner surface of the bottle mouth and the stopper. If the shape of the cross section 19 of the triangle to be cut is large, the water seal water 1
8a increases, the removal of water at the time of opening the plug is troublesome, and further deepening the gap impedes the sealing mechanism of the plug.
2, and the non-contact portion 20 with the stopper from the tip 23 of the mouth of the bottle
Is preferably 2 to 5 mm.

Next, FIG. 17 is an enlarged view of the upper portion 11 of the contact between the mouth of the bottle and the stopper, and has a structure in which the tip of the shoulder 22 of the stopper is lower than the tip 23 of the mouth of the bottle. That is, it has a structure in which the stopper is inserted deeply. By doing so, the water seal water 18b can be formed on the upper part of the shoulder. If the end of the shoulder 22 is deeply lowered and the non-contact portion 21 with the mouth of the bottle is made long, the sealing mechanism of the stopper is obstructed. Therefore, the length of the non-contact 21 is determined in consideration of the water sealing state. Is the tip of the mouth of the bottle 2
A structure lowered by 1 to 2 mm from 3 is preferable. Na, shoulder of stopper 2
With the gradient of 2, the amount of water sealed can be adjusted. That is, if the gradient is reduced, the amount of water sealed is increased, and if the gradient is reduced, the amount of water is reduced.

FIG. 5 shows a mechanism for adjusting the temperature of the air in the tank instead of adjusting the temperature of water. In place of the water temperature control / water circulation device 8 of the device described with reference to FIG.
A W heater 25 and a cooler 28 of 20 Kcal / Hr are installed on the side wall, and a temperature control device 27 is
Is used to adjust the temperature of the air in the tank. In the case of a room temperature of 25 ° C., the temperature in the tank became 20 ° C. in about 15 minutes.

FIG. 6 shows an example of the use condition of a culture tank of a water temperature control system using a large heat-retaining air tank 29. The size of the tank is 200cm in width, 110cm in height, 90c in depth
m. The walls and doors have a thermal insulation structure. Inside, there are eight spray ports with a spray angle of about 90 degrees and a reach of about 60 cm on the ceiling and four on each side wall, almost equally installed. The floor is a perforated board. There is a water storage section 15 at the lower part. The constant temperature water is circulated by the external water temperature control / water circulation device 8 and sprayed indoors.
A water-permeable basket 32 containing 24 culture bottles
12 cases each with 3 cases on the water-permeable shelf 33 of the step
The cases are stacked, the cart is stopped from being sprayed with water, and the door 30 is opened and closed to put in and out. Stop water spraying when moving the cart in and out. The number of the culture bottles to be stored is 1152, which allows cultivation of an average of 76 samples every day for 5 days of BOD measurement. If the case storage capacity of the cart is increased, the number of carts is increased, and the inner volume of the culture tank is increased, the number of culture bottles can be easily increased. Since the water storage unit 15 is installed at the bottom of the tank, the floor inside the tank is higher than the external floor, so that the inclined plate 34 for taking in and out the cart is installed.
By stopping water about 15 minutes before removing the trolley, dripping from the trolley is greatly reduced. In addition, it is possible to reciprocate from the thermostat to the measuring desk while the culture bottle is loaded on the cart.

Since the present invention is configured as described above, the following effects are exhibited.

Since a normal culture bottle without a collar can be used, there is no need to use a culture bottle with a glass collar that is easily damaged, and the work of injecting sealing water into the color of the culture bottle with a collar and opening the bottle after the culture is completed. In addition, there is no need to perform the operation of removing the water, which is more advantageous than using a culture bottle with a collar in terms of work efficiency.

Compared with the method of immersing the culture bottle in the constant temperature water bath and culturing, the labor for removing the culture bottle from the thermal insulation tank is reduced. The use of a large heat-retaining air tank has the advantage that a large number of culture bottles can be easily taken in and out using a cart, and the transportation can be handled as it is. In addition, the wetness of the floor around the handling is greatly improved. Further, since the amount of water used is extremely small as compared with the thermostatic water bath, the rise up to the thermostat can be performed quickly. In addition, since water becomes dirty as it is used, it is necessary to exchange water.However, the method of the present invention is easier because the amount of water is extremely small compared to immersion culture. On the other hand, the constant temperature water tank can not be deepened when taking out and putting in the culture bottle, and the tank must be expanded horizontally.In contrast, in the case of the present invention, the culture bottle can be stacked in the upper direction using the shelf. Since the tank can be expanded upward, there is an advantage that the installation area is smaller than that of the water tank.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view of a culture tank and an explanatory view of a water seal.

FIG. 2 is a plan view of a culture tank with a lid removed.

FIG. 3 is a cross-sectional view for explaining a culture bottle with a collar.

FIG. 4 is a partially enlarged view of a water sealing void structure above a contact between a mouth and a stopper of a culture bottle.

FIG. 5 is a longitudinal sectional view of a culture tank for controlling air temperature.

FIG. 6 is a diagram showing the use of a large culture tank.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Culture bottle 2 Glass color 3 Desorption color 4 Culture bottle with color 5 Water 6 Insulated air tank 7 Lid 8 Water temperature control and water circulation device 9 Circulation piping 10 Water spray port 11 Contact upper part of bottle mouth and stopper 12 Enlarged view 13 Water Sealing water 14 Perforated disk 15 Water storage unit 16, 17 Enlarged view 18a, 18b Water sealing water 19 Triangular cross section 20, 21 Non-contact part 22 Shoulder of stopper 23 Tip of bottle mouth 24 Water circulation device 25 Heater 26 Sensor 27 Temperature control device 28 cooler 29 large heat-retaining air tank 30 door 31 trolley 32 basket 33 shelf 34 inclined plate 35 hole

Claims (4)

[Claims] A culture bottle (1) for colorless BOD measurement is stored in a light-insulated heat-retaining air tank (6) to control water temperature.
A constant temperature water is continuously or intermittently sprayed by a water circulation device (8) to keep the inside of the tank at a constant temperature, and a water seal water (13) is placed on a contact upper portion (11) of a mouth and a stopper of a culture bottle (1). A method of constant temperature culturing and the structure of a culture tank while preventing evaporation of water around the stopper of the culture bottle (1) and preventing oxygen in the air from entering the inside of the culture bottle (1). . 2. The structure of a warm air tank wherein a water circulation device (24) is installed instead of the water temperature control / water circulation device (8) in claim 1, and the temperature of the air in the tank is controlled by the temperature control device. 2. The method for isothermal culturing according to 1. 3. A stopper formed by cutting off the shoulder of the stopper of the culture bottle (1) in the shape of a triangular cross section 19 having a cross-sectional area of 10 to 15 mm 2 and forming a non-contact portion (20) between the stopper and the mouth of the bottle. ) At a depth of 2 to 5 mm from the tip (23) of the mouth of the bottle, and water sealing water (18a) around the upper portion of the mouth of the culture bottle.
The structure of the culture bottle having a valley space in which the water accumulates, and the culture method according to claim 1 or 2, wherein the culture bottle is used. 4. The end of the stopper shoulder (22) of the culture bottle (1) is located 1-2 mm from the tip (23) of the mouth of the bottle,
The structure of a culture bottle (1) having a structure in which a water seal water (18b) accumulates on an upper portion of a stopper shoulder (22), and using the same.
Or the culture method according to claim 2.