JP4808100B2 - Evaluation method of bacterial coaggregation ability - Google Patents

Description

  The present invention relates to a method for evaluating the co-aggregation ability of bacteria, particularly oral bacteria present in the oral cavity, an external force application device for capsules for observation in liquid used therein, a holder for fixing capsules for observation in liquid, and efficacy agents It relates to the evaluation method.

  For the purpose of preventing oral diseases such as bad breath, periodontal disease, and cavity, various oral compositions for cleaning plaque and tongue coating in the oral cavity are provided. These oral compositions include those containing a proper amount in the mouth, such as mouthwashes, those that clean the mouth using auxiliary tools such as toothbrushes, such as dentifrices, and denture cleaning agents And the like that are washed outside the oral cavity after desorption.

  In recent years, as a mechanism of the occurrence of plaque and tongue coating, bacteria such as general bacteria, pathogenic bacteria, and Fusobacterium bacteria existing in the oral cavity coaggregate to form aggregates, and the coaggregates adhere to oral tissues・ It has been found to be caused by proliferation. For this reason, it is important to accurately evaluate the coaggregation ability of such bacteria by dispersing the oral bacterial coaggregation mass on the tissue surface or saliva, or suppressing the growth of bacteria after adhesion. Important for development.

  Conventionally, in order to grasp the coaggregation ability of such bacteria, a coaggregation reaction is caused in a test tube having a sample stage at the bottom, and the generated coaggregation mass is allowed to settle on the sample stage. This was done by observing the attached co-agglomerates using a laser confocal microscope or the like. For this reason, the coaggregation lump could be observed only with optical resolution, and the coaggregation ability could not be grasped based on its fine shape and the like.

  On the other hand, a technique that enables electron microscope observation of a wet or liquid sample is provided (see Patent Document 1 below).

US Pat. No. 6,992,300

  The present invention has been made in view of the above-mentioned problems, and is a method for evaluating bacterial coaggregation ability that can accurately and objectively and easily perform bacterial coaggregation ability, and for in-liquid observation used therein. The present invention provides a capsule external force application apparatus, a capsule fixing holder for observation in liquid, and a method for evaluating a coagglomerate dispersant.

  The present inventor considered that the coaggregation ability of bacteria can be objectively and easily understood by using the above-described technique, and has completed the present invention.

  The present invention applies an external force to the capsule for observation in liquid containing two or more kinds of bacteria to be evaluated, and observes the co-aggregation state of the bacteria before and after applying the external force with a scanning electron microscope, The object is achieved by providing a method for evaluating the coaggregation ability of bacteria, which evaluates the coaggregation ability of the bacteria based on the observation result.

  Further, the present invention is an external force application device for a capsule for observation in liquid used in the method for evaluating the coaggregation ability of the bacterium of the present invention, which is attached to an observation stage of a scanning electron microscope and is observed in the liquid An external force applying device for an in-liquid observation capsule, comprising: a fixing holder that accommodates and fixes the capsule for use, and a vibration source that vibrates the fixing holder.

  Further, the present invention is a fixing holder for accommodating and fixing a capsule for observation in liquid used in the method for evaluating the coaggregation ability of bacteria of the present invention, which is attached to an observation stage of a scanning electron microscope A submerged observation capsule fixing holder comprising positioning means for positioning the submerged observation capsule at a predetermined position of the fixing holder according to the form of the submerged observation capsule. To do.

  The present invention also relates to a method for evaluating an effect agent using the above-described method for evaluating the coaggregation ability of a bacterium according to the present invention, wherein the capsule for observation in liquid includes a coaggregant lump dispersing agent together with the bacterium. In addition, external force is applied to the capsule for observation in liquid, and the co-aggregation state of the bacterium is observed with a scanning electron microscope before and after the external force is applied, and the efficacy of the dispersant is evaluated based on the observation result A method for evaluating a co-agglomerated dispersant is provided.

  According to the present invention, a bacterial coaggregation ability evaluation method capable of accurately, objectively and easily assessing bacterial coaggregation ability, an external force application device for a submerged observation capsule used therein, and submerged observation A capsule fixing holder and a method for evaluating a co-agglomerate dispersant are provided.

  The present invention will be described below based on preferred embodiments thereof.

  First, an external force application device (hereinafter also referred to as an external force application device) of a capsule for observation in liquid (hereinafter also referred to as a capsule) used in the method for evaluating the coaggregation ability of bacteria of the present invention will be described.

FIG. 1 is a schematic view showing an embodiment of the external force application device of the present invention.
As shown in FIG. 1, an external force application device 1 is an external force application device for a capsule 11 used in a method for evaluating the co-aggregation ability of bacteria according to the present invention, which will be described later. A holder 2 and a vibration source 3 that vibrates the fixing holder 2 are provided. In this embodiment, the fixing holder 2 is attached to the observation stage 10 of the scanning electron microscope via a vibration propagation plate 31 that transmits the vibration of the vibration source 3.

  As shown in FIG. 2, the capsule 11 is an instrument for storing a sample containing bacteria to be evaluated, and the upper cylinder 12, the ultrathin film 13 that closes the upper end opening of the upper cylinder 12, and the upper cylinder 12 are moved up and down. Are provided with a seal member 14 for forming a partition sample enclosure and a lower cylinder 15. A spiral (not shown) that is screwed to each other is provided at a lower portion of the inner peripheral surface of the upper tube 12 and an upper portion of the outer peripheral surface of the lower tube 15, and the upper tube 12 and the lower tube 15 are screwed together. Thus, the sample can be sealed. A pair of protruding pieces 120 and 150 projecting outward are provided on the outer peripheral surface portions of the upper cylinder 12 and the lower cylinder 15. A projection 151 that protrudes downward is provided at the center of the bottom surface portion of the lower cylinder 15. As the capsule 11, specifically, a wet SEM capsule (trade name QX-capsule) manufactured by Quantomics, Israel is preferably used.

  The fixing holder 2 is a jig that accommodates and fixes the capsule 11 and has a bottomed cylindrical shape. The fixing holder 2 includes positioning means for positioning the capsule 11 at a predetermined position of the fixing holder 2 according to the form of the capsule 11. In this embodiment, the positioning means is configured by a recess 21 provided at the upper end of the fixing holder 2 so as to correspond to the protruding pieces 120 and 150 of the capsule 11. By fitting the projecting pieces 120 and 150 into the concave portion 21, the capsule 11 can be accurately positioned with respect to the fixing holder 2. Even when the capsule 11 is once removed from the fixing holder 2 and accommodated again, the same positioning is performed. Can be accommodated in a state. A fitting hole 22 into which the protrusion 151 of the lower cylinder 15 of the capsule 11 is fitted is provided in the center of the inner bottom portion of the fixing holder 2. A spiral hole 23 is provided on the lower surface of the fixing holder.

  The fixing holder 2 is a holder provided to function also as a cooling means for the capsule 11. That is, the fixing holder 2 is fixed by screwing the spiral hole 23 into the male spiral 40 of the cooling device 4 made of a Peltier element fixed to the observation stage 10. At this time, the male spiral 40 is inserted into the insertion hole 32 of the vibration propagation plate 31, and the vibration propagation plate 31 is interposed between the fixing holder 2 and the cooling device 4. With such a configuration, the temperature rise in the capsule 11 due to the irradiation of the electron beam is suppressed, and vibration from the vibration source 3 is transmitted to the capsule 11. The fixed holder 2 is made of an aluminum alloy, and the vibration propagation plate 31 is made of a nonmagnetic stainless alloy.

  The vibration source 3 only needs to have an output capable of applying vibration to the capsule 11 via the vibration propagation plate 31 and the fixing holder 2. As the vibration source 3, an ultrasonic vibrator, a piezoelectric element, or the like can be used. The vibration frequency of the vibration source is preferably 30 kHz to 55 kHz in consideration of the resonance frequency of the ultrasonic transducer.

  Next, the method for evaluating the coaggregation ability of bacteria of the present invention will be described based on a preferred embodiment applied to the evaluation of the coaggregation ability of oral bacteria.

  First, the external force application device 1 is set on a scanning electron microscope (not shown). That is, the observation stage is removed from the main body of the scanning electron microscope, the fixing holder 2 is attached to the removed observation stage (not shown) via the vibration propagation plate 31, and the vibration source is attached to the vibration propagation plate 31. 3 is attached so that the vibration of the vibration source 3 can be propagated to the fixing holder 2 via the vibration propagation plate 31.

  Next, a sample containing oral bacteria that are bacteria to be evaluated is enclosed in the capsule 11. Here, as the sample containing oral bacteria, two or more strains selected from standard strains and clinical isolates belonging to the genera Fusobacterium, Streptcoccus, Actinomyces, Porphyromonas, Prevotella, etc. are used. Alternatively, a biological sample such as tongue coating may be used. To enclose the sample, the upper tube 12 is turned upside down and the sample is attached to the ultrathin film 13, and then the optical density (OD: Optical Density) at a wavelength of 600 nm is measured as a control of the number of cells, and OD = 0 Adjust the bacterial cell concentration to be 0.05 to 0.5. Diluents used for adjusting the bacterial cell concentration include physiological saline, physiological saline phosphate buffer (pH 7.4), physiological in addition to the sample imaging buffer provided by Quantomics. A salt-added Tris-HCl buffer (pH 8) or the like can be used. Since the coaggregation reaction is promoted by divalent cations, physiological saline or physiologically salted Tris-HCl buffer (pH 8) containing 0.1 mM calcium ions and magnesium ions is particularly preferable. In order to observe with good contrast, general electron staining agents such as uranyl acetate, lead acetate, and phosphotungstic acid can also be used. In this case, it is preferable to use an electronic stain at an aqueous solution concentration of 0.1 to 2 wt%.

  Next, the capsule 11 enclosing the sample is accommodated in the fixing holder 2. At this time, the protrusion 151 is fitted into the fitting hole 22, and the protruding pieces 120 and 150 are fitted into the recess 21 to be positioned.

  Next, an observation stage is attached to the main body of the scanning electron microscope, the state of the bacterial coaggregation mass contained in the sample is observed with the scanning electron microscope, and the bacterial coaggregation state in a specific observation field is photographed. To do. The acceleration voltage during observation is preferably 1-30 kV, and the detection mode is preferably a backscattered electron detection mode or a secondary electron detection mode.

  Next, the external force application device 1 is operated to transmit the vibration of the vibration source 3 to the capsule 11 via the vibration propagation plate 31 and the fixing holder 2, and an external force is applied to the capsule 11. The time for applying the external force to the capsule 11 by vibration is set according to the bacteria to be evaluated, the configuration of the external force application device, and the like. In the case of the present embodiment, considering that the vibration source 3 applies vibration to the capsule 11 via the vibration propagation plate 31 and the fixing holder 2, the vibration time is preferably 10 seconds to 5 minutes, and 30 seconds. 2 minutes is more preferable.

  Next, the application of external force due to vibration is stopped, and the coaggregation state of the bacteria after the external force is applied is observed in the same observation field as before the external force is applied, and the coaggregation state of the bacteria is imaged.

  And based on this observation result (observation image), the coaggregation ability of the said bacteria before and after applying external force by vibration is evaluated. Specifically, the number of cells forming the coaggregate is counted, and if there is no change in the number before and after, the binding force or adhesion between the bacteria forming the coaggregate is not observed. If the adhesion between the membrane and bacteria is evaluated as strong and the number is reduced, the binding force between the bacteria forming the coaggregate or the attached membrane and bacteria It is evaluated that the adhesive force between is weak. Instead of counting the number of bacterial cells, the observation result can be analyzed by a binarization process or the like with an image analysis device or the like, and similarly evaluated based on the result.

  Thus, according to the method for evaluating the coaggregation ability of bacteria using the external force application device 1 of the present embodiment, it is possible to observe the coaggregation state of bacteria before and after applying an external force in the same observation field. Therefore, it is possible to evaluate the coaggregation ability of bacteria accurately, objectively and easily.

  Next, the method for evaluating a coaggregate dispersing agent according to the present invention will be described based on a preferred embodiment applied to the evaluation of an oral composition using the method for evaluating the coaggregation ability of bacteria.

  The method for evaluating the coaggregant lump dispersant according to this embodiment is the same as the method for evaluating the coaggregation ability of bacteria according to the above embodiment, except that the dispersant is added to the sample.

  Examples of the co-agglomerate dispersant include dispersants such as a vehicle component contained in the oral composition shown below. Specifically, sugars such as galactose, N-acetylgalactosamine, lactose, lactitol, maltitol, palatinit, sorbitol, mannitol, xylitol, erythritol, and other sugar alcohols, sodium lauryl sulfate, lauroyl sarcosine sodium, lauroyl methyl taurine sodium , Polyoxyethylene hydrogenated castor oil, polyoxysorbitan fatty acid ester, sucrose fatty acid ester, ralylglycoside, lauroylglutamic acid, and the like. The concentration of the dispersant is set according to the type of the dispersant, the concentration of the dispersant for which the effect is to be evaluated, and the like. The dispersant applied to the present invention is not limited to these dispersants, and other dispersants can be widely applied.

  According to the method for evaluating the coaggregate dispersing agent of the present embodiment, the coaggregation state of the bacteria before and after applying the external force can be observed in the same observation field. The effect of the dispersant can be accurately, objectively and easily performed.

The present invention is not limited to the embodiment.
In the method for evaluating the co-aggregation ability of bacteria in the embodiment, an external force is applied to the submerged observation capsule in the electron microscope. Before applying the external force, the submerged observation capsule is temporarily removed from the scanning electron microscope. After taking out and applying external force to the in-liquid observation capsule, the co-aggregation ability of bacteria can be evaluated by placing the in-liquid observation capsule again in the scanning electron microscope and observing it. . In this case, like the holder for fixing the capsule for observation in liquid of the embodiment, since it is provided with positioning means for uniquely fixing the capsule for observation in liquid to the holder for fixation, it is added before applying external force. It is possible to observe the co-aggregation state of the bacteria after the same observation field.

  In the method for evaluating the co-aggregation ability of bacteria in the above embodiment, the time for applying the external force is set according to the bacteria to be evaluated, the configuration of the external force application device, etc., but the time to apply the external force instead of the time is shortened and the number of times You may control the external force applied by. Furthermore, taking an image every time an external force is applied, taking a necessary number of images (for example, 10 to 500 times), and regenerating them continuously (moving images) to dynamically evaluate the aggregating ability of bacteria. It becomes possible. Since the evaluation includes the concept of time, it is possible to evaluate the process that changes by applying external force.

  The present invention is suitable for evaluating the coaggregation ability of oral bacteria as in the above embodiment, but besides this, coaggregation at the time of forming a composite biofilm by general bacteria or mold formed in a humid environment It can also be applied to performance evaluation.

  In addition, the present invention is suitable for evaluating the efficacy of the coagglomerate dispersant contained in the oral composition as in the above-described embodiment. It can also be applied to the efficacy of fungicides and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples. The scope of the present invention is not limited to such examples.

[Example 1]
Using the following electron microscope, capsule for observation in liquid, and external force application device, observe the coaggregation state of the bacteria before and after applying external force for the following samples including the following oral bacteria, and based on the observation results, Aggregation ability (anti-dispersion ability) was evaluated as follows. An electron micrograph is shown to Fig.2 (a) and (b).

Scanning electron microscope: Hitachi, S-4300SE / N
Capsule for observation in liquid: QX-capsule QX-102, manufactured by Quantomics
External force application device: Vibration source (ultrasonic vibrator, maximum output 100W, resonance frequency 45KHz), aluminum alloy fixing holder

<Sample>
Oral bacteria: Fusobacterium nucleatum ssp. Polymorphum ATCC10953 strain, Streptococcus sobrinus B13 strain Dispersion medium: 0.1 mM calcium chloride, 0.1 mM magnesium chloride, 0.15 M sodium chloride-added Tris-HCl buffer Electronic stain: 0.35 wt% aqueous solution Concentration of uranyl acetate

<Observation conditions>
The acceleration voltage of the electron microscope was 15 kV and observed with a backscattered electron detector.

<External force application conditions>
With the external force application device, a vibration of 45 kHz was applied for 1 minute.

<Evaluation of co-aggregation ability>
Based on the observation results of the bacterial coaggregation state before and after applying external force, the coaggregation ability was evaluated by counting the number of cells observed in each.

  As shown in FIGS. 2 (a) and 2 (b), the coaggregation ability of oral bacteria subjected to the evaluation was found to be sufficiently strong because there was almost no change in the number of cells observed with external force applied. It was.

[Example 2]
The efficacy of the dispersant was evaluated as follows in the same manner as in Example 1 except that 1% by mass of galactose was added to the sample as a dispersant for the aggregate. The observation results are shown in FIGS. 3 (a) and 3 (b).

<Evaluation of efficacy of efficacy agent>
Based on the observation result of the co-aggregation state of bacteria before and after applying external force, the effect is that the number of cells observed is clearly reduced compared to the case without dispersant (Fig. 1). The efficacy of the agent was evaluated.

  As shown in FIGS. 3 (a) and 3 (b), it is clear that the number of bacterial cells is reduced by galactose, and there is an ability to reduce the coaggregation ability of bacteria or an ability to disperse bacteria. all right.

Example 3
The efficacy of the dispersant was evaluated in the same manner as in Example 1 except that 1% by mass of erythritol was added as a dispersant to the sample. The observation results are shown in FIGS. 4 (a) and 4 (b).

  As shown in FIGS. 4 (a) and 4 (b), it is clear that the number of bacterial cells is decreased by erythritol, and it can be seen that the number is further decreased as compared with galactose. Thus, it was found that erythritol has the ability to reduce the coaggregation ability of bacteria or to disperse bacteria.

It is a mimetic diagram showing a section of one embodiment of an external force application device used in the evaluation method of the co-aggregation ability of bacteria of the present invention. It is a figure which shows the scanning electron micrograph which shows the observation result by the Example of the evaluation method of the coaggregation ability of the bacteria of this invention, (a) before applying external force, (b) is observation after applying external force It is a photograph which shows a result. It is a figure which shows the scanning electron micrograph which shows the observation result by the Example of the evaluation method of the coagglomerate dispersing agent of this invention, (a) before applying external force, (b) after applying external force It is a photograph which shows an observation result. It is a figure which shows the scanning electron micrograph which shows the observation result by the Example of the evaluation method of the coagglomerate dispersing agent of this invention, (a) before applying external force, (b) after applying external force It is a photograph which shows an observation result.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 External force application apparatus of capsule for observation in liquid 2 Holder for fixing capsule for observation in liquid 3 Vibration source 10 Stage for observation 11 Capsule for observation in liquid

Claims (9)

  Apply external force to the capsule for observation in liquid containing two or more kinds of bacteria to be evaluated, observe the co-aggregation state of the bacteria before and after applying external force with a scanning electron microscope, and based on the observation results And a method for evaluating the coaggregation ability of the bacteria.   The method for evaluating the coaggregation ability of bacteria according to claim 1, wherein an external force is applied to the capsule for observation in liquid in the scanning electron microscope.   Before applying an external force, the capsule for observation in liquid is once taken out of the scanning electron microscope, and after applying an external force to the capsule for observation in liquid, the capsule for observation in liquid is again put in the scanning electron microscope. The method for evaluating the coaggregation ability of bacteria according to claim 1, wherein the coaggregation ability is observed. Evaluation method of co-aggregation ability of the bacterium according to any one of claims 1 to 3 applying an external force to vibrate the liquid for observation capsules. Evaluation method of co-aggregation ability of the bacterium according to any one of claims 1 to 4 wherein the bacterium is oral bacteria.   A device for applying an external force to a capsule for observation in liquid used in the method for evaluating the coaggregation ability of bacteria according to claim 2, wherein the device is applied to an observation stage of a scanning electron microscope and contains the capsule for observation in liquid A device for applying an external force to a capsule for observation in liquid, comprising: a fixing holder that is fixed by being fixed; and a vibration source that vibrates the fixing holder. A fixing holder for accommodating and fixing a capsule for observation in liquid used in the method for evaluating the coaggregation ability of bacteria according to claim 3 ,
Used attached to an observation stage of a scanning electron microscope ,
Positioning means for positioning the capsule for observation in liquid at a predetermined position of the fixing holder according to the form of the capsule for observation in liquid is provided , and the positioning means is provided on the outer peripheral surface portion of the capsule for observation in liquid It consists of a recess provided corresponding to the protruding piece, and
A holder for fixing a capsule for observation in liquid, which can be fixed by being screwed to a cooling device fixed to the observation stage . A method for evaluating a coaggregate dispersing agent using the method for evaluating the coaggregation ability of bacteria according to claim 1,
A dispersion agent is included in the in-liquid observation capsule together with the bacteria, and external force is applied to the in-liquid observation capsule, and the co-aggregation state of the bacteria before and after applying the external force is measured by a scanning electron microscope. A method for evaluating a co-agglomerate dispersant, which is observed and the efficacy of the dispersant is evaluated based on the observation result.   The method for evaluating a co-agglomerate dispersant according to claim 8, wherein the co-agglomerate dispersant is a component that can be blended in an oral composition.