JPH02124099A - Measurement of living bacterium number - Google Patents

Abstract

PURPOSE:To estimate the number of gram negative bacteria in a specimen and thereby rapidly, simply, specifically measure the number of living bacteria in a high sensitivity for examining the state of infection, etc., by measuring an optical change caused by a gelling reaction of the specimen with the extracted solution of a limulus hemocyte component. CONSTITUTION:A specimen is reacted with a solution in the presence of a water-soluble polysaccharide or/and a water-soluble derivative thereof containing beta-1,3-glucan as a constituent, the solution being prepared by dissolving the freeze-dried product of a hemocyte component extract solution originated from a limulus of the family Limulus in a 0.1M tris-hydrochloric acid buffer solution (pH7.3). An optical change based on the resultant gelling reaction is measured with a nepherometer, etc., and the number of living gram-negative bacteria in the specimen is estimated from the extent of the measured optical change, thereby measuring the number of the living gram-negative bacteria in the specimen.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention is useful in the field of clinical testing and sanitary testing to determine the degree of infection of the human body by microorganisms and the degree of contamination of food and the environment by microorganisms. The present invention relates to a method for measuring the number of viable bacteria that has been implemented for this purpose.

[Background of the Invention Co-bacteriological testing is used in the field of clinical and sanitary testing.
For example, for testing the state of bacterial infection, including sifting bacterial groups that infect the human body, testing for bacterial contamination in food, testing water quality of wastewater, sewage, pools, etc., and testing the state of contamination of food and the environment. It is widely implemented as a test to find out. Among these, the measurement of the number of viable bacteria in a test sample has been widely used for a long time as a screening test to determine the degree of infection or contamination by bacteria 'a'.

Viable bacteria count measurements include, for example, Staphylococcus bacteria and Streptococcus.

Jj+li It is carried out targeting many bacteria such as Gram-positive bacteria such as Jj+li, Diphtheria bacterium, and Mycobacterium tuberculosis, and Gram-negative bacteria such as Escherichia coli, Vibrio, Neisseria, and Pseudomonas aeruginosa. 1 For example, by observing the smear-stained specimen of the test sample under a microscope and counting the number of viable bacteria, or by measuring the turbidity of the test sample using a spectrophotometer, the number of viable bacteria in the test sample can be determined. There are two methods to estimate the number of live bacteria in the test sample, such as a method to directly measure the test sample, and a method to estimate the number of colonies on the culture medium after culturing the test sample in an appropriate medium F. A typical example is a method for estimating the number of cells (cultivation method).

Among these methods, in the method of directly measuring the test sample, it is difficult to detect unless the number of viable bacteria in the test sample is about 106 cells/ml or more, and in the method using a smear-stained specimen, However, there are problems such as the need for skill in staining the bacteria, and the culture method requires 24 to 72 hours to obtain results, and there are some bacteria that are difficult to culture. There are problems in that the method cannot be applied to all bacteria, and there has been a desire to develop a method for measuring the number of viable bacteria that can be measured with high sensitivity in a short time and is easy to operate.

[Purpose of the Invention] The present invention was made in view of the above-mentioned situation, and an object thereof is to provide a method for measuring the number of viable bacteria that can perform highly sensitive measurements in a short time and is easy to operate.

[Configuration of the Invention] The present invention provides a test sample and horseshoe crab blood cell components (hereinafter referred to as AL).
It is abbreviated as ) Extract liquid (hereinafter abbreviated as AL warm solution)
Gram-negative bacteria characterized by measuring the optical change based on the gelation reaction that occurs when reacting with and estimating the viable number of Gram-negative bacteria in the test sample from the degree of the optical change. This invention is a method for measuring the number of viable bacteria.

That is, the present inventors mainly investigated the cell surface of Gram-negative bacteria.
It is a type of ribopolysaccharide present in Py
Endotoxin (also known as rogen)
Hereinafter, it will be abbreviated as ET. ), the method utilizes the phenomenon in which the ＼L solution is activated by ET and coagulates.
Noting that the so-called limulus test is widely used due to its simplicity and low cost, we thought that by utilizing this phenomenon, it would be possible to detect Gram-negative beaches as viable bacteria. As a result of our research, we found that the AL warm solution also reacts with live Gram-negative bacteria to produce a gelation reaction, and the optical change based on this gelation reaction was measured, and based on the degree of the optical change, it was possible to determine the The present inventors have discovered that it is possible to estimate the number of viable Gram-negative bacteria in a sample, and have completed the present invention.

To carry out the measuring method of the present invention, for example, it may be carried out as follows.

That is, first, the test sample and the AL hot solution are thoroughly mixed and reacted, and the optical change based on the gelation reaction that occurs as a result of the reaction is measured. Next, the degree of optical change obtained here is expressed as a relationship between the number of viable Gram-negative bacteria and the degree of optical change, obtained in advance using a solution containing a predetermined number of Gram-negative bacteria as a test sample. By applying it to the calibration curve, the number of viable Gram-negative bacteria in the test sample can be estimated.

The AL hot solution that can be used in the present invention includes Limulus (1, imulus), Tachypheus
Heus) genus or Carcinoscorbius (Carci)
It is extracted from the blood cells of horseshoe crabs belonging to the genus Noscorpius), and is not particularly limited as long as it causes a coagulation reaction by reaction with ET.

Kei, for example, ACC (ASSOCIATES OF)
CAPE C0D) company.

Of course, it is also possible to use products prepared based on freeze-dried AL warm solutions commercially available from Hemakem, MAB, Mallinckrodt, and others.

The test sample for the measurement method of the present invention is not particularly limited as long as it is considered to contain viable Gram-negative bacteria, but examples include blood, serum,
Examples include biological body fluids such as cerebrospinal fluid and urine, drinks such as juice and milk, extracts from foods such as kamaboko and fish paste, and living environment water such as tap water, pool water, and sewage.

The measurement method of the present invention uses an AL warm solution, except that it uses a test sample that is thought to contain live Gram-negative bacteria, and uses an optical method based on the gelation reaction that occurs as a result of the reaction with ET. gi of ET by measuring the change
This can be carried out according to the per se known ET measurement method for determining f1, and the other reagents used are also according to the reagents used in the per se known ETIJ standard method as described above.
They may be selected and used as appropriate. Using a warm AL solution, the optical change due to the gelation reaction resulting from the reaction with ET is expressed as Fl
! ! ET is measured by determining I.
Examples of the T gil + standard method include the nephelometric method, which measures the turbidity that occurs with gelation, and the gelation time measurement method, which measures the time until the turbidity that occurs with gelation reaches a certain value. It will be done.

It goes without saying that the measuring method of the present invention can employ any of these methods such as turbidimetry and gelling time measuring method, but especially the gelling time measuring method, in particular the test sample and AL hot solution. A gelation time measuring method described in JP-A-61-159162, which involves irradiating a reaction solution containing light with light and measuring the time until the amount of transmitted light of the reaction solution decreases by a predetermined percentage from the initial amount of transmitted light after the reaction has started. is more preferably used.

In the measurement method of the present invention, the pH at which the AL warm solution test sample is reacted can be any pH at which the factor that reacts with ET in the AL solution and causes a coagulation reaction is not inactivated. However, the range of 6 to 8 is usually preferably used. In addition, in order to avoid effects caused by the pH of the test sample and always perform measurements within this PI range, tris(hydroxymethyl)aminomethane (Tris) must be
It is desirable to coexist with FJ agents that are commonly used in the field of biochemistry, such as s) and Goods' Buffer. In addition, the temperature at which the AL warm solution test sample is reacted may be any temperature that does not deactivate the factors that react with ET in the AL solution and cause a coagulation reaction, but usually 0 to 40°C. More preferably 25-40
°C is used. The measuring method of the present invention can be carried out using, for example, a toxinometer ET-201 (Wako Pure Chemical Industries C Ltd. ffi).
, it can be carried out using a specialized device for turbidimetric time analysis such as LAL-5000 (manufactured by ACC), or it can be similarly carried out using a measuring device using other optical principles such as a spectrophotometer. Can be implemented.

By the way, it is generally known that AL hot solution ET reacts with not only ET but also carboxymethylated β-1,3-glucan and coagulates.
l,, Iliochem, Biophys, R
e5earch Communicatjon, evil■
Engineering η-, 434-439 (1981)]. In addition, this phenomenon is caused by β-1,3-glucan (β-1,3-glucan) coexisting in the AL solution.
Hereinafter, it will be abbreviated as GL. )! It is already known that the 3-receptivity factor is induced by reacting with GL or its derivatives [Iwanaga et al., Japanese Journal of Mycology.

31 Sea (□l, 78]-803 (1983)
Ko .

Therefore, such substances, namely GL and GL
If there is a possibility that derivatives may be contained, it is necessary to take measures to prevent this.

In such cases, the inventors first found out.

The measuring method of the present invention may be carried out using the following method for which a patent application has been filed (Japanese Patent Application No. 63-45069). That is, when reacting the test sample with the horseshoe crab blood cell extract, a water-soluble polysaccharide (hereinafter referred to as
It is abbreviated as GLPS. ) or/and its water-soluble derivative in large quantities and carry out the method of the present invention, GLF
Since the S-susceptibility factor is inactivated and the AL hot solution coagulation reaction by GL does not occur, only the coagulation reaction due to ET occurs, so if this phenomenon is used, even if the OL is contained in the test sample as a component of the membrane. It is possible to specifically detect only gram-negative bacteria even if there are a mixture of bacteria including gram-negative bacteria.

GLPS and its water-soluble derivatives that can be used in this measurement method are not particularly limited as long as they are polysaccharides that contain OL as a constituent and are water-soluble. However, if it falls down, various bacteria (e.g. Alcaljgenes, Lamjn
Natural polysaccharides obtained from the cell walls of A. aria [, Agrobacl: erium [, etc.], yeasts (e.g., Saccharomyces genus, etc.), mushrooms (e.g., Shiitake, Suehirotake, Kawaratake, etc.), specifically, for example, curdlan, Pachyman, sclerokun, lentinan, schizophyllan, coriolan, etc.

Alternatively, storage polysaccharides of algae (for example, brown algae, euglenoid algae, diatoms, etc.), specifically, for example, laminaran.

Paramylon, etc., or these can be prepared using conventional methods, for example, Large Organic Chemistry Vol. 19, 7th edition, pages 70-lot, supervised by Mujio Kotake,
IO day, May 1962, morning'f! ;Bookstore; A, E
, C1, Arke et al., Physiochemistr.
y, 175-188 (1967); T
.

5asaki et al., Europe, J. Cancer,
15. Carboxymethyl group according to the method described in 211-215 (1967) etc.

Carboxyethyl group, methyl group, hydroxyethyl group,
Preferred examples include water-soluble derivatives obtained by introducing a hydroxypropyl group, a sulfopropyl group, etc., and these may be used alone or in an appropriate combination of two or more.

Examples of methods for allowing GLPS or/and its water-soluble derivatives to coexist during the reaction with the AL warm solution test sample include:
Dissolve these in water, buffer solution, dilute alkaline solution, etc.
A method of dissolving a freeze-dried AL product using this method, adding GLPS prepared by the method described above or/and its Method of adding a solution of a water-soluble derivative, G
A method of adding LPS or/and its water-soluble derivative to a sample. A reagent obtained by freeze-drying a warm AL solution to which a required amount of GLPS or/and its water-soluble derivative has been added in advance is added to distilled water for injection. Examples include a method of preparing by dissolving in a buffer solution, etc. However, no matter which method is used, when the AL warm solution test sample reacts, the GL-sensitive factors coexisting in the AL solution are inactivated, but ET and IE are inactivated.
T A GLPS that does not inhibit the reaction with sensitive factors and the coagulation reaction of the A L solution caused by that reaction.
and/or its water-soluble derivative coexist, and is not limited to these methods.

The concentration of GLPS or/and its water-soluble derivative in the reaction solution is, for example, a lyophilized product of an AL solution or a lyophilized product of an AL solution.
Detection sensitivity for hot solution manufacturing loft and ET (EIJ/m
Although there is some variation depending on factors such as l).

Usually, in the reaction solution of the test sample and AL warm solution, 10
Preferably about 0 ng/ml to 100 B/ml,
10μ.

Examples include a concentration range of approximately 10 +ng/ml to 10 +ng/ml.

However, when the above-mentioned commercially available lyophilized AL product was prepared as an AL warm solution for ET measurement, the temperature was 0.03
It has a detection sensitivity for ET of ~5 EU/ml, and a coagulation reaction is caused by adding 0.1 to 1000 ng/ml of a polysaccharide having GL as a constituent or/and its derivative. When such an AL hot solution is used, the concentration of GLPS and/or its water-soluble derivative to be added is 100%, which is the concentration that causes a coagulation reaction by the polysaccharide containing OL as a constituent component and/or its derivative.
It is desirable that it be 0 times or more.

The test method of the present invention is carried out by allowing GLPS or/and its water-soluble derivative to coexist during the reaction between the test sample and the AL warm solution.
This may be carried out in accordance with the method for measuring the viable count of Gram-negative bacteria according to the present invention using the AL warm solution described above, except for allowing a predetermined amount of LPS or/and its water-soluble derivative to coexist. .

The present invention will be described in more detail with reference to Reference Examples and Examples below, but the present invention is not limited by these in any way.

[Example Reference Example 1. Preparation of carboxymethylated curdlan 540 ml of toluene and 30 ml of ethanol e were added to 60 g of curdlan (manufactured by Wako Tsumugi Kogyo Co., Ltd.), 61 g of a 50% aqueous sodium hydroxide solution was added dropwise thereto, and the mixture was heated to 50°C. The mixture was stirred for 1 hour. To this was added a solution of 35 g of monochloroacetic acid dissolved in 100 ml of a mixed solvent of toluene ditanol = 9:1, and the mixture was further stirred at 50°C for 1 hour. Further add a sodium hydroxide aqueous solution and a monochlorobutyric acid solution to this reaction solution.
After repeating twice, it was cooled and stored overnight. This is 9
The mixture was poured into 0% methanol 11, and the resulting precipitate was collected and dried to obtain 142 g of 1 fll crystal. The obtained crude crystals were dissolved in 1,420 mouths of distilled water, and the p of this solution was
H was adjusted to 8 using dilute hydrochloric acid. To this, 12.78 ml of methanol was added dropwise while stirring, the resulting precipitate was collected, washed with 500 ml of 90% methanol, dried, and the desired carboxymethyl curdlan (hereinafter referred to as CMCU) was collected.
It is abbreviated as ) was obtained.

Example 1 (test sample) The bacterial suspension shown below was used as a test sample.

Enterococcus faecalis (clinical isolate nigerum positive 1a), S. faecalis
occus aureus ATCC25923(
Gram positive+? j), Escherichia c.
oli ATCC25922 (Gram-negative bacteria), Kl
ebsiella pneumoniae (m-bed isolate nigerum-negative bacteria) Pseudomonas aer
ugin.

sa ATCC27853 (Gram-negative bacteria) was cultured on a uQ slant at 37° C. for 18 hours, and the resulting bacterial cells were suspended in sterile physiological saline so that the turbidity was the same as McFarland 0.5. These solutions were used as stock solutions, and the appropriate serial dilutions were used as test samples.

The number of viable bacteria in the stock solution was measured by a conventional method using a trypto soy agar plate medium, and based on the results, the number of viable bacteria in each test sample was determined.

(Measurement procedure) A freeze-dried product of a blood cell component extract derived from horseshoe crabs of the genus Limulus (hereinafter abbreviated as LAL, manufactured by Wako Tsumugi Pharmaceutical Co., Ltd., coagulation sensitivity: 0.03 EU/ml, for 5 mi) was subjected to ET. 0.1'l Tris-W acid buffer (pH 7,3
) Add 0.1 ml of the test sample to 0.1 ml of the solution (hereinafter abbreviated as LAL solution) dissolved in 5.0 ml,
After mixing well, use a toxin meter ET-201 (manufactured by Wako Tsumugi Kogyo Co., Ltd.) to measure 11fl at 37°C until the amount of transmitted light of the sample decreases by 5% (hereinafter referred to as gelling time 1).
It is called 18. ) was measured.

(Results) The results are shown in Figure 1. In addition, in Figure 1, 〇- is Es
The results obtained with the test sample containing Eherichia coli ATCC25922 as the bacterium, -.
udomonas acruginosa ATCC27
853 as a bacterium.
is or 5taphyrococcus aureus
The results obtained using test samples containing ATCC25923 as bacteria are shown.

As is clear from this result, the Gram-negative bacterium Esc.
hcrichiacoli, Pseudomonas
aeruginosa.

For KICbsjella pneumoniae, the logarithm of the number of viable bacteria] α and the logarithm of 110 at the time of gelation are 11
A good correlation was observed for 0.

On the other hand, the Gram-positive bacterium Enserococcus
f'aecalis and Sl; aphyrococcu
s aurcus shows a 5% decrease in the amount of transmitted light of the sample within cjO minutes for any number of births, +1Q at the time of Kelization.
It was not possible to estimate the number of viable bacteria.

From the above results, when the test sample was gelled with L A L 78 liquid using Toxin Meter ET-201, 111
1 S! It can be seen that when this assay is carried out, Gram-positive bacteria in the sample are not detected, and only the number of viable Gram-negative bacteria can be specifically measured.

Example 2 (Test test N) The same material as in Example 1 was used.

($11 constant operation method) LAL (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd., coagulation sensitivity: 0.03
ELI/ml, for 5ml) in Reference Example 1, ? I! ! I
Manufactured CMCU 1. 0.1M Tris containing Omg
- A solution dissolved in 5.0 ml of hydrochloric acid buffer (pl+7.3) (does not contain endotoxin) (hereinafter referred to as CMCU)
-Abbreviated as LAL solution.

) After adding 0.1 ml of the test sample to 0.1 ml and mixing well, the gelation time was measured in the same manner as in Example 1.

(Results) The results are shown in Figure 2. In addition, in Figure 2, 〇- is Es
cherjchia colj ATCC25922 as a bacterium, -△- indicates Pseudomonas aeruginosa AT
The results obtained from the test sample containing CC27853 as a bacterium are expressed as -low is Enterococcus faec
alis or S siaphyrococcus aur
The results obtained with the test sample containing eus8rCC5923 as a bacterium are shown.

As is clear from this result, even when the CMCU-LAL solution was used for the determination of E.
scherichia colj, Pseudomo
For nas aeruginosa, a good correlation was observed between the logarithm of the number of viable bacteria and the logarithm of gelation time (to 1), but for Enterococcus, a gram-positive bacterium,
S faecalis and Sl: aphyrococc
For any viable bacterial count, the amount of transmitted light of the sample did not show a 5% decrease within 60 minutes, making it impossible to estimate the viable bacterial count based on the gelation time.

From the above two results, even when the gelation time of the test sample was measured using the CMCU-LAL solution using a toxin meter ET-201-, Gram-positive bacteria were not detected in the sample, and Gram-negative bacteria were detected. It can be seen that only the number of viable bacteria can be specifically measured.

Example 3. Measuring the number of viable fungi (test, sample) The bacterial suspension shown below was used as a test sample.

Aspcrgillus niger IFO4407
(mold), Aspergi11us jerreus
IFO4100 (mold), 85 perzillus
flavus IFO5839 (mold), 5poro
thrix 5chenckii IFO8158 (mold) + Cryptococcus albindu
s IFO1320 (yeast), Candida a
lbicans IFOO579 (yeast), Candi
da t, ropjcalis IFO1400 (yeast) - Candidakrusei IFOO584 (
yeast), Candida albicans IF
O1060 (yeast) was slant cultured at 30° C. for 2 days, and the resulting bacterial cells were suspended in an appropriate amount of lJi bacterial saline to prepare both suspensions.

The number of viable bacteria in each suspension was determined by a conventional method using a modified Bennett method using an agar plate medium.

(AL solution) LAL solution used in Example 1 and C used in Example 2
MCU-LAL solution was used.

(Measurement operation method) The same procedure as in Example 1 was used.

(Results) Table 1 shows the gelation time (Tg) obtained for each test sample. Note that Table 1 also shows the number of viable bacteria in each test sample.

Table 1 As2゜ Aspergillus.

Can,=Candjda.

As is clear from Jingo's results, conventional LAL solutions react with some fungi such as mold and yeast, but C
It can be seen that the MCU-LAL solution does not react with fungi, that is, the CMCU-LAL solution specifically reacts with Gram-negative bacteria.

[Effects of the Invention] As mentioned above, the present invention can be performed in a shorter time with a simpler operation compared to the conventional viable bacteria count testing methods such as methods using smear-stained specimens and culture methods. Moreover, it provides a method that can specifically measure the number of viable Gram-negative bacteria in a test sample with high sensitivity, and is a great invention that contributes to this industry.

[Brief explanation of drawings]

Figure 1 shows the calibration curve obtained in Example 1, where the horizontal axis is the number of viable bacteria (cells/ml) and the vertical axis is the time (minutes) until the amount of transmitted light decreases by 5% (hereinafter referred to as (abbreviated as gelation time)
are shown respectively. In the figure, -〇- is Escherichia
The results obtained from the test sample containing E. coli ATCC25922 as bacteria are shown in the table below.
-△- indicates the results obtained from the test sample containing P. pneumoniae as the bacteria.
The results obtained from the test sample containing S. eruginosa ATCC27853 as a bacterium are
coccus faecalis or S shyaphyro
The results obtained using test samples containing coccus aureus ATCC25923 as bacteria are shown. Figure 2 shows the standard curve obtained in Example 2, where the horizontal axis represents the number of viable bacteria (cells/ml) and the horizontal axis represents the number of viable bacteria (117 cells/ml) at the time of gelation.
1 is shown respectively. In the figure, -〇- is Escherichia
-Δ- indicates the results obtained from the test sample containing E.coli ATCC25922 as a bacterium.
-low is En En
terococcus faecalis or 5jap
hyrococcus aureus ATCC259
The results obtained using test samples containing 23 as bacteria are shown. Patent Applicant: Wako Pure Chemical Industries, Ltd. Figure 10'10'' 105 Ki Fungi Takajo 5 (Ik/m1) Figure Procedural Amendment January 3, 1990

Claims (2)

[Claims] (1) Measure the optical change based on the gelation reaction that occurs when the test sample and horseshoe crab blood cell extract are reacted, and determine whether the Gram-negative bacteria in the test sample is viable based on the degree of the optical change. A method for measuring the viable count of Gram-negative bacteria, characterized by estimating the number. (2) Claim 1 in which a water-soluble polysaccharide containing β-1,3-glucan and/or a water-soluble derivative thereof is coexisting when reacting the test sample with the horseshoe crab blood cell extract. Measurement method described in.