JPH0582901B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for measuring the number or amount of microorganisms, and particularly to a method suitable for measuring a non-uniform microorganism suspension.

[Conventional technology]

When preparing bacterial solutions for a variety of purposes, such as investigating the growth status of bacteria, animal experiments, and vaccine production, it is extremely often necessary to know the number and amount of bacteria.
Conventionally, a method for measuring such microorganisms is as described in Japanese Patent Application Laid-Open No. 140151/1983, in which microorganisms labeled with fluorescence are irradiated with ultraviolet light using an antigen-antibody reaction, and the amount of excitation light generated is measured electrically. A similar method is to quantify microorganisms by measuring the amount of microorganisms, or as a similar example, as disclosed in JP-A-57-144995, microorganisms and other solid substances are centrifuged immediately before fluorescence intensity measurement. Furthermore, as described in JP-A-58-17598, the fluorescence intensity of umbelliferon was measured after treating the bacteria with an organic solvent such as toluene. Conventional measurement examples exist.

Additionally, as a method for measuring without using a special fluorometer as mentioned above, there is a long-known method that measures turbidity by measuring transmitted light and scattered light, and calculates the concentration of bacteria from this. It has been widely used as a method.

[Problem that the invention seeks to solve]

Among the conventional techniques mentioned above, in the method of measuring fluorescence intensity using a fluorescent marker etc., ¹⁰³ to 104 ^CEU /
Highly sensitive measurement is expected as it is possible to measure the number of nl bacteria, but a special centrifugation operation is required, and growth is observed over time while continuing to culture for drug susceptibility tests and other purposes. The problem is that it cannot be applied as is when it is necessary to perform a measurement, and when a fluorescently labeled microorganism is brought into contact with a fluorescently labeled antibody to obtain a fluorescently labeled microorganism, the microorganism must be fixed (killed), and culture cannot be continued after measurement. In addition, in the method of pretreatment with an organic solvent, the bacterial cell membrane is destroyed by toluene, and the esterase contained within the bacterial cell can sufficiently hydrolyze the umbelliferone derivative, allowing highly sensitive bacterial measurements. However, since the bacteria themselves are lysed and killed, there are problems in that not only can they be returned to the culture system to observe changes over time, but they also cannot be used as samples with known bacterial concentrations for other experiments. .

On the other hand, with the method of measuring microorganisms using spectrophotometry, it is possible to continue culturing, but the detection sensitivity is low.
The problem is that it can only be detected in a bacterial suspension with a high concentration of 10 ⁶ to 10 ^{7 CFU} /ml.

When the cell suspension is dilute, the intensity of light scattered by the cell suspension corresponds to the total number of cells. Therefore, measurement using scattered light is possible. However, this is true under the condition that the sample has a constant turbidity, that is, the bacteria to be tested is uniform. Therefore, if the shape and size of bacteria change significantly depending on the stage of growth, the turbidity and the number of bacteria may not necessarily be in parallel. Therefore, during the logarithmic growth phase, the intensity of scattered light corresponds to the total number of bacteria, so even if it is possible to measure accurately, for example, staphylococci may form clusters like clusters of grapes under certain conditions during the stationary phase. There is a problem in that it may not be able to be successfully applied to bacterial count measurements when the bacteria undergo filamentation, such as when the antibiotic cephalexin is applied to bacteria, and the morphology of the bacteria changes drastically.

Further, the photometry of the transmitted light and scattered light is performed in one direction, either vertically or horizontally, depending on the device. The photometric direction is generally regulated by the shape of the cell; for example, when using a general glass or quartz square cell or test tube, it is horizontal, and when using a 96-well microplate, it is naturally vertical. (vertical direction).
However, the form of growth varies depending on the type of bacteria, and depending on the form of growth, problems may arise in measurement depending on the direction of photometry.

That is, in the case of homogeneous bacteria such as E. coli, measurement can be performed in both the horizontal and vertical (up and down) directions. However, as mentioned earlier, staphylococci and streptococci grow into clusters as they multiply.
Alternatively, they may form chains and therefore tend to settle in the cell. In this case, a problem arises in horizontal photometry in that the light beam passes through the supernatant and the correct number of bacteria is not reflected. Furthermore, green coli bacteria are known to form a bacterial film on the surface of bacterial liquid, but when photometrically measured in the vertical (up and down) direction, all of the light flux is reflected, making measurement impossible.

The purpose of the present invention is to be able to measure the microorganisms in the sample without killing them, to be able to accurately measure even if the morphology of the bacteria changes during the measurement and is no longer homogeneous, and to be able to measure various bacterial species that have different growth forms. The purpose of this invention is to provide a method for measuring microorganisms that allows measurement to be performed without error using the same device.

[Means to solve the problem]

As shown in Figure 5, the above purpose is to irradiate light from both the horizontal and vertical (up and down) directions, and after passing through the bacterial suspension, which is the sample, detect both the transmitted light and the scattered light. This is achieved by comprehensively evaluating the obtained information and measuring the number of bacteria by correlating it with the number of bacteria.

[Effect]

The light irradiated onto the bacterial suspension of the sample changes the amount of transmitted light (absorbance) or the scattering intensity depending on the bacterial concentration, that is, the number of microorganisms. Now, in the case of bacteria that grow in different ways, 1. They divide uniformly,
2. As the bacteria divide and multiply, they become cloudy and at the same time form a bacterial film on the surface of the bacterial liquid. 3. They are arranged like bunches of grapes or chained together into strings, forming clumps. Let's consider three cases in which something is created, grows larger, and sinks. Furthermore, consider the following two growth states of each bacterium. In other words, 1. The state from the lag phase in preparation for division to the logarithmic growth phase in which 2 divisions occur actively. This state is a relatively dilute bacterial suspension.

2 The state from the logarithmic growth phase to the stationary phase, where an equilibrium is maintained between new bacteria and dying bacteria, and the number of viable bacteria appears to be constant. Growth is saturated in a closed culture medium, and the growth pattern is unique to the bacterial species. The bacterial solution has a fairly high concentration, ranging from about 10 ^{7 CFU} /ml to about 10 ^{9 CFU} /ml, which is the maximum that can be grown in vitro.

A total of 6 types of bacteria and 2 types of growth states.
Assume the case of a street. In such a bacterial suspension, the fifth
As shown in the figure, light is irradiated from the horizontal and vertical directions, and after the light passes through the bacterial suspension, both transmitted light and scattered light are detected. The signal from each detector is
Next we will explain how it works with the data.
Further, schematic diagrams of the state of the bacterial liquid and photometry are shown in Figs. 6, 7, and 8.

1 In the case of a homogeneous bacterial solution 1-1 Induction phase to logarithmic phase: Only scattered light provides correct data in both the horizontal and vertical directions, and data cannot be obtained from transmitted light due to lack of sensitivity.

1-2 Logarithmic phase to stationary phase: Both scattered light and transmitted light can be used as data in both horizontal and vertical directions. (Figure 6) 2. When applying a bacterial film 2-1 Lag phase to logarithmic phase: Due to the low concentration of the bacterial solution, only scattered light is data. Since the bacterial membrane has not formed at this point, the value is also correct in the vertical direction.

2-2 Logarithmic phase to stationary phase: Due to the formation of a fungal film, it is difficult for light to pass through when irradiated from the vertical (up and down) direction (Figure 7). This results in incorrect data for both scattering and transmission. Scattered light is correctly detected when irradiated from the horizontal direction. Also,
At this point, the bacterial solution is highly concentrated, so the induced light can also be detected correctly.

3 In the case of sedimenting bacteria 3-1 Induction phase to logarithmic phase: Transmitted light is not detected due to low concentration bacterial solution. In addition, since they have not yet grown to the point where they form clumps, scattered light can be detected correctly in both vertical and horizontal directions.

3-2 Logarithmic phase to stationary phase: At this point, division and
Proliferation progresses, forming bacterial clumps that tend to settle. Therefore, both scattered light and transmitted light are measured correctly in the vertical direction, but in the horizontal direction, the light flux may pass through the supernatant, and does not reflect the accurate number of bacteria. (FIG. 8) Therefore, both the scattered light and the transmitted light result in erroneous data.

The above can be summarized as shown in Figure 4.

In this way, correct data cannot be obtained with only data from one detector, and the number of bacteria cannot be accurately measured. By performing a comprehensive evaluation using all four data from four detectors, it is possible to measure the entire growth process, which changes in various ways, and various bacterial species with different growth forms, without error, using the same device. .

〔Example〕

Examples of the present invention will be described below.

Example 1 FIG. 1 is a diagram showing the growth of P. aeruginosa (Pseudomonas aeruginosa) monitored. The horizontal axis is the culture time after inoculating the bacteria into the medium, and the vertical axis is the scattering intensity (○ and ● marks) and absorbance (△ and ▲ marks), respectively.

When bacteria are inoculated into a culture medium, they first enter a lag phase.
During this period, the inoculated bacteria prepares for division and multiplication, and in order to adapt to the new environment, the bacteria produce enzymes, coenzymes,
Physiological growth occurs to store essential substances. The length of this lag period varies greatly depending on the age of the inoculum and the composition of the medium. In the case shown in FIG. 1, this corresponds to 3 to 4 hours. In this range, there are no noticeable differences due to the direction of irradiation, scattered light, or transmitted light. After passing through the lag phase, the bacterium rapidly begins to divide into two and enters the logarithmic growth phase, which repeats division into two regularly at a constant rate. 1st
In the figure, it is seen after 4 hours, but until around 5.5 hours it is early and the concentration of bacteria is still small, so scattered light is detected and growth is observed, but it is not observed in transmitted light. From around the 6th hour, it becomes possible to detect even transmitted light. However, once the proliferation has progressed sufficiently, Pseudomonas aeruginosa has the property of forming a bacterial membrane on the surface of the bacterial liquid. In the case shown in the figure, bacterial film formation begins around 8 hours. For this reason, when irradiating from the vertical direction, the variation in measured values increases, and as the proliferation progresses further, erroneous data will be output for both scattered light and transmitted light.
As for the state of bacteria, nutrients in the culture medium decrease, waste products accumulate, and pH changes progress, and a balance is maintained between new bacteria and dying bacteria, and the number of viable bacteria is constant. Continue.

As mentioned above, relatively early after vaccination, the first
In the figure, data a and b become valid, followed by c and d, but eventually b and d become erroneous data and cannot be used as data. So a, b, c, d
It is extremely important to collect all the information and ultimately perform a comprehensive evaluation, so that the number of bacteria can be accurately measured.

Example 2 Figure 2 shows the monitoring of the growth of S. aureus. In the case of this figure, the induction period appears to be about 2 hours, and there is no significant difference in the data from the four detectors during this period. After that, as proliferation progresses, scattered light becomes detectable around 2.5 hours and transmitted light becomes detectable around 3.5 hours. Accordingly
Within the range of 2.5 to 3.5 hours, scattered light from both horizontal and vertical directions is used as data, and transmitted light is considered to indicate no bacterial growth. As staphylococci multiply, they become arranged in clusters of grapes. The reason why a bacterial agglomeration that started from the division of one bacteria shows this arrangement is because the first division plane and the next division plane are perpendicular to each other, but
This is due to the progression of separation of daughter cells during division, which begins at one end of the septum and extends to the other end, resulting in an irregular arrangement. The clusters of bacteria arranged in clusters of grapes gradually become larger and tend to settle in the liquid medium. When light is irradiated horizontally on a bacterial solution in which sedimentation has occurred, the light beam will pass through the supernatant. Such a state appears in the horizontal direction in FIG. 2 after the 9th hour of scattered light. The growth curve shows a decreasing trend and the number of bacteria is erroneously measured to be lower than the actual number. In the vertical direction, the correct number of bacteria was detected, indicating that the growth was saturated (○-○ and △-△).

As mentioned above, in the case of bacterial groups that tend to form clumps, such as the above-mentioned staphylococci and streptococci that only divide in one direction and are connected in a string-like manner, early after inoculation, Data a and b are valid, followed by data c and d. When sufficient multiplication occurs, a and c become incorrect data. In Example 1, a and c were correct data, but in this case they are incorrect data. This indicates that the types of valid data differ depending on the bacterial species.

Example 3 Figure 3 is an example of monitoring the growth of Escherichia coli. As in Examples 1 and 2, the signal from the detector for transmitted light shows a delay of about 3 hours compared to the signal for scattered light. Therefore, data a and b from the scattered light detector are valid regardless of the direction during a relatively early period. However, as time passes and the proliferation progresses, in the case of E. coli, the bacterial liquid becomes uniformly cloudy. Therefore, as shown in FIG. 3, transmitted light becomes effective after 5 to 6 hours, and moreover, it becomes effective in both horizontal and vertical directions. After all, Escherichia coli is an example in which either a or b is valid data in the early period, and a, b, c, and d are all valid data after proliferation has progressed.

〔Effect of the invention〕

According to the present invention, measurement can be performed while continuing culture without damaging the sample. Furthermore, even if the sample loses uniform turbidity during the measurement, accurate measurement can be continued with the same device. Furthermore, for various microorganisms that proliferate and take on different forms, light irradiated from two directions, vertically and horizontally, is transmitted light and scattered light, respectively, and transmitted light and/or scattered light change according to changes in form. By selecting the scattered light, the number of bacteria can be accurately measured from the intensity of the selected transmitted light and/or scattered light.

[Brief explanation of drawings]

Figures 1 to 3 are data obtained by the microbial measurement method according to the present invention, Figure 4 is data shown by the state of the bacterial liquid and photometry, and Figure 5 is the data obtained by the microbial measurement method according to the present invention. The configuration diagram of the means for implementing the method, and FIGS. 6 to 8 are schematic diagrams showing the state of the bacterial liquid and photometry. 1, 2... Light source, 3... Cell, 4... Bacterial liquid (sample), 5, 6... Scattered light detector, 7, 8...
...Detector for transmitted light, 9...Microorganisms, 10...Bacteria film, 11...Clump of microorganisms.

Claims (1)

[Claims] 1. Irradiating a sample solution containing microorganisms to be cultured with light,
In the method of optically measuring the number of microorganisms, light is irradiated from two directions, vertical and horizontal, and the intensity of each transmitted light and scattered light is measured over time. For microorganisms that grow homogeneously, select horizontal scattered light intensity data or vertical scattered light intensity data as growth phase data from the four intensity data of transmitted light and scattered light during the growth phase. Calculate the number of bacteria from , and use one of the following as stationary period data: horizontally scattered light intensity data, horizontally transmitted light intensity data, vertically scattered light intensity data, or vertically transmitted light intensity data. For microorganisms that form a bacterial film on the sample liquid surface, select horizontal scattered light intensity data or vertical scattered light intensity data as growth phase data and calculate the number of bacteria from this intensity data. Calculate the number of bacteria from the data, select horizontal scattered light intensity data or horizontal transmitted light intensity data as stationary phase data, calculate the number of bacteria from this intensity data, and calculate the number of bacteria that settles to the bottom of the sample solution. selects horizontal scattered light intensity data or vertical scattered light intensity data as growth phase data, calculates the number of bacteria from this intensity data, and selects vertical scattered light intensity data or vertical scattered light intensity data as stationary phase data. A microorganism measurement method characterized by selecting intensity data of transmitted light in a direction and calculating the number of bacteria from this intensity data.