JPH0227260A - Bacteria identifying and counting method - Google Patents

Abstract

PURPOSE:To count the number of colonies without missing by culturing bacteria for a proper time and scanning the cultured bacteria by a laser spot having a sufficiently small diameter to perform sampling and judging the shape and size of each colony on the basis of the obtained data. CONSTITUTION:The bacteria on a membrane filter are cultured for a proper time and a colony grown into a semispherical shape having a diameter of about several times is set as an object to be counted. The diameter of a laser beam spot is made sufficiently small as compared with the colony and the scanning of an object to be examined of the colony or foreign matter is performed by said laser beam spot to sample data. With respect to the data belonging to the same object to be examined, the size L=(Xm, Ym) (Xm and Ym are the data lengths in scanning directions X, Y), oblateness e=Xm/Ym and area ratio r=N/XmYm (N is the total number of the sampling data belonging to the same object to be examined are calculated and one wherein those values are respectively within definite ranges is judged to be a colony to perform counting.

Description

Detailed Description of the Invention [Industrial Field of Application] This invention is a method for counting microorganisms contained in a so-called fluid, such as a liquid or gas, in which microorganisms are distinguished from other foreign substances and only microorganisms are counted. The present invention relates to a discriminative counting method for

[Prior Art] Microorganisms contained in beverages such as drinking water and beer are generally useless and are sometimes harmful, so measuring their number is essential for quality control.

For example, if yeast 1:h remains in beer, it will proliferate and impair quality, so yeast is removed using a filter at the time of bottling, and a microbial test using a culture method is performed before shipping. Yeast counts are tested over time. - There are various methods for detecting and counting microorganisms in liquid, but since the yeast contained in beer before shipping is minute and few in number, it is necessary to have the ability to detect them individually.

! - As a method suitable for the above, there is ``Counting method of microorganisms'' in Japanese Patent Application Laid-Open No. 63-53447. The gist of this application is to dye the microorganisms filtered and captured by a membrane filter with an appropriate fluorescent dye, irradiate this with laser light, detect and count the fluorescence emitted from the microorganisms, and to carry out the method. In embodiments, the S/N ratio of the microorganism's fluorescence is improved by selectively detecting the fluorescence using a photomultiplier tube and by making the spot diameter of the laser beam substantially similar to the size of the microorganism. It is intended to improve FIG. 6 shows the configuration of the above-mentioned conventional microorganism counting method, in which the laser light from the laser light source 1 is swept by a scanning mirror 2, focused by a focusing lens 3, and placed on an XY child table. The membrane filter 4 is scanned. 6
is the drive mechanism for the XY child table. Fluorescence emitted by microorganisms passes through a cut filter 7 and a condensing lens 8, and is received by a photomultiplier tube 9. The control section 10 controls the operation of each section and processes the received light signal, and the recorder 11 records the signal for the detected microorganism.

[Problem to be solved] According to the results of applying the microorganism counting method according to the above application to beer before delivery, beer contains a considerable number of foreign substances in addition to yeast, and the foreign substances also emit fluorescence. As a result, it was discovered that all the samples were counted without distinguishing between yeast and yeast, resulting in a large error. Therefore, distinguish between yeast and foreign substances.
It is necessary to count the

The present invention has been made in view of the above, and aims to provide a discrimination counting method for distinguishing between microorganisms such as yeast and people and counting only microorganisms by augmenting the microorganism counting method disclosed in the application described in -. 1 target.

[Step F for solving the problem] This invention involves filtering a fluid sample containing microorganisms through a membrane filter, staining the captured microorganisms with a fluorescent dye, scanning the membrane filter surface with a laser beam, This is a discrimination counting method that distinguishes microorganisms from other foreign substances and counts them in a microorganism counting method that detects and counts the fluorescence emitted by microorganisms.

1- The microorganisms on the membrane filter described above are cultured for an appropriate time and counted as colonies that have grown into a spherical shape with a diameter at least several times larger than the size before culture. On the other hand, the diameter of the laser beam spont is sufficiently small compared to the colony, and the colony or foreign object is scanned and sampled. For the data belonging to the same subject, calculate the subject's size L1 platysma ratio e and area ratio r, and if these are within a certain range, the subject is determined to be a colony. Determine that there is one and count it.

In the sampling data belonging to the same subject described in 1-, the data lengths in the scanning direction (X) and the direction perpendicular to the scanning (Y) are respectively Xra and Ym, and the total number of sampling data belonging to the same subject is N, The oblateness ratio 01 area ratio r and the size L of the subject are calculated using the following formulas.

Flat/V-rate: e =:Xm/Ym ・
Old (I) Area ratio: r = N/Xm Ym
...(2) Size: l, = (Xs,
Y+s) −” (3) For these glues, e and r, k+++ < e < I/km −
”(4) r>kr...
...(5)ks<L<kL
. . . The shape and size of the object are determined using the conditional expression (6).

[Operation] In the method for identification and counting of microorganisms according to the present invention, colonies whose +i'f diameter has grown at least several times as large after culturing for an appropriate time are counted.

Figure 1 shows the growth behavior of draft beer yeast by culture. Individual yeasts captured on membrane filters initially have a diameter of 6 μm.
The colony is approximately elliptical in size, but if cultured using a membrane filter under appropriate conditions such as nutrition and temperature, it will become a hemispherical colony with a larger diameter over time.

However, although there are individual variations in the diameter of the colonies,
The grains are concentrated in a relatively narrow range indicated by the curves ■ and ■ shown in the figure, that is, the grains are uniform. Looking at the center curve ■, it is only 7 μm in 12 hours, but 10 μm in 24 hours.
The colony grows to approximately 0 μm (approximately 15 times the original size). The colony that has grown in this way is stained with a fluorescent dye and is irradiated with laser light. The size is much larger than the initial yeast, In addition, since it is hemispherical, it is expected that detection will be extremely easy. However, in practice, how much to grow requires consideration in consideration of the required time. The counting method is based on the assumption that there is no time for culturing in order to avoid delays in shipping, and therefore, even in this case, the shorter the culturing time, the better.Therefore, in this invention, for example, the culturing time is about 20 hours. By culturing, the diameter is expanded several to ten times.

FIG. 2 explains the scanning of the laser spot in this invention, in which the pitch interval d' in the Y direction is
is scanned in the X direction and sampled at a time interval Δt equal to N d'.

Here, since the sampling data is detected in a distributed manner for all of the subjects, first, data belonging to the same subject are grouped together. This will be explained with reference to FIGS. 3(a) and 3(b).

In figure (a), for any data p, if at least one data is detected at any of the eight adjacent positions qt-'qa, these are considered to be continuous, and each By searching the data p for continuous data, a data group belonging to the same subject is changed and stored.

Figure (b) shows examples (a) and (b) of summarized data groups.

Next, since the shape of the colony is 16 spheres, it is determined whether the data distribution is circular.

Figure 4 explains the shape determination method, and Figure (a)
In the data group belonging to the same subject indicated by the * mark, the data lengths in the X and Y directions are respectively Xm and Ym, and when these are equal or approximate, one condition of a circle is satisfied. The degree of approximation was determined by calculating the flatness e of the equation (km,
1/km). In figure (a), Xm=E3. Ym=7, e
:t, t'7. However, e alone cannot make it circular. In figure (b), data groups (c) and (d) both have Xm:4. Although YI11=4,
(D) has a shape close to a rectangle. Therefore, the area ratio r in equation (2) is calculated, and the determination is made using equation (5). In this case, the number of data N is 12 in (c) and 8 in (d), and the area ratio r is 12/IG:0.75.8/16=
It is 0.5. If the sampling density is t.min, then r=π/4:o,'ya for a perfect circle, so an appropriate kr is set to -t.

From this, it is determined that the foreign object is circular or approximately circular; however, circular foreign objects may still be present. As mentioned above, the size of the colony is almost constant, so for the size scale in equation (3), kS in equation (6),
Check by kL.

ks and kL are determined by the culture time. As a result, even if they are circular, abnormally large or small objects are not recognized as colonies. However, it is inevitable that foreign objects of the same size as a colony will be judged as 1, but the number of such objects is small.

[Example 5] Fig. 5 shows a flowchart of the processing procedure in the method for identification and counting of microorganisms according to the present invention, in which sampling is performed by scanning the laser spot on the object, and the detection signal from the sampling is contains low-level noise, so check the peak value and remove it ■. ■The detection signal is stored in memory as digital data.

Addresses in the memory are stored in correspondence with sampling positions, and this is performed for the entire surface of the membrane filter.

After such sampling is completed for the entire surface of the membrane filter, in step (3), according to the order of memory addresses FY, it is searched for data at each position to see if data is detected in eight adjacent locations, and If there is at least one, it is assumed that they are continuous, and the results are stored in memory.

Next, in ■ and ■, by equations (+) and (2),
i,; e and the area ratio r are calculated, and the shape is determined using formulas (4) and (5). For those that meet these conditions, the size L according to formula (3) is determined by formula ( 6), and only colonies of a certain size are counted as colonies. Items that do not meet the conditions in ■, ■, and ■ are considered foreign substances■.

In the above, the order of determining the shape and size can be changed, but in practice, it is better to first determine the shape and then determine the size of the circular object, including foreign objects. This method is efficient because it avoids having to judge many subjects.

In the following, setting the scanning pitch of the laser spot, checking the peak value (■), processing the continuity judgment process (■), and determining the shape and size (■ to ■) can all be easily performed using normal techniques. Therefore, detailed explanation will be omitted. In addition, the method described in [U] is applied to the method for identification and counting of microorganisms related to the application originally explained, and the basics and Section 11, such as scanning a membrane filter with a laser beam, receiving and counting fluorescence, are based on that method. The same is true.

In the embodiments, a liquid such as beer is described as an example, but the present invention can be used not only for liquids but also for gases, and can be applied to so-called fluid samples in general.

[Effects of the Invention] As is clear from the above explanation, in the method for identifying and counting microorganisms according to the present invention, microorganisms captured by a membrane filter are cultured for an appropriate time to form a hemispherical colony with a large diameter, In contrast, scanning sampling is performed using a laser spot with a sufficiently small diameter, and the shape and size are determined based on the data, and those that do not meet the conditions are considered foreign objects, making it possible to accurately count most colonies without missing them. . It can be applied not only to beer but also to any case where yeast culture is allowed, and the technology for counting microorganisms contained in the liquid has a great effect.

[Brief explanation of the drawing]

Fig. 1 is a curve diagram showing the growth of colonies against the passage of incubation time for beer yeast. Fig. 2 is a curve diagram showing the growth of colonies with respect to the elapse of incubation time for beer yeast. 3(a) and (b) are explanatory diagrams of the relationship, scanning pitch, and sampling interval,
FIGS. 4(a) and 4(b) are explanatory diagrams of a method for determining data groups belonging to the same specimen in the microorganism identification and counting method according to the present invention. An explanatory diagram of the shape determination method, FIG. 5 is a flowchart of the process-L order in an embodiment of the microorganism identification and counting method according to the present invention, and FIG. 6 is a block diagram of the conventional microorganism counting method. DESCRIPTION OF SYMBOLS 1... Cable light source, 2... Scanning mirror 3... Focusing lens, 4... Membrane filter, 5... XY child table 6... XY drive mechanism, 7... Cut filter, 8 ···Condenser lens,
9... Photomultiplier tube, 10... Control unit, 11...
Recorder, ■～■...Flowchart number. Figure 1 □ Jealousy 4 hours (hunting) Figure 3 (a) (b) Figure (a) (b) j Xrn-4-1 Figure 5 ○

Claims (1)

[Claims] (1) A fluid sample containing microorganisms is filtered through a membrane filter, the captured microorganisms on the membrane filter are stained with a fluorescent dye, and the surface of the membrane filter is scanned with a laser beam to emit radiation from the microorganisms. In the microorganism counting method that detects and counts fluorescence, the microorganisms on the membrane filter are cultured for an appropriate period of time, and colonies that have grown into a hemispherical shape with a diameter at least several times the size before culture are targeted. The diameter of the spot of the laser beam is set to be sufficiently small compared to the colony, and the colony or foreign object is scanned and sampled, and based on the continuity of each sampled data, it is determined that the sample belongs to the same object. For the determined data, determine the oblateness e and area ratio r for determining the shape of the subject, and the size L, and on the condition that e, r, and L are each within a certain range, A method for identification and counting of microorganisms, characterized in that the specimen is determined to be the above-mentioned colony and then counted.