JP2002034595A - Method for living cell detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting whole living cells including gram- negative bacteria in a logarithmic growth phase by solving a problem that gram-negative bacteria in a logarithmic growth phase cannot be dyed, which is a defect in a conventional method for living cell detection. SOLUTION: This method for living cell detection is characterized in that a cell-containing medium is dyed with a fluorescent enzyme substrate in the presence of a calcium ion and fluorescence is assayed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting living cells, and more particularly to a method for specifically detecting living cells by staining a medium containing cells with a fluorescent enzyme substrate in the presence of calcium ions. . The present invention also relates to a reagent kit for use in the above method, and a live cell detection device for performing the above method.

[0002]

2. Description of the Related Art Detection of living cells in a medium is one of very important means for confirming a sterilized state and detecting abnormalities in the survival state of cells. As a method for specifically detecting live cells, a vital staining method (Darzynkiewicz, Z. et al.,
Experimental Cell Research, 95 , 143-153 (1975))
A method in which a fluorescent enzyme substrate or dye is added to a medium and the fluorescence emitted in cells existing in the medium is measured (Lundgr
en, B et al., Oikos, 36, 17-22 (1981)).
In addition, the inventors disclosed in JP-A-10-179191 that a fluorescent enzyme substrate capable of changing into a pH-dependent fluorescent substance in living cells was added to a medium, and the detection was performed using the pH of the fluorescent substance.
A method for detecting a living cell, which is performed by irradiating excitation light suitable for, or JP-A-10-215894,
After adding a fluorescent enzyme substrate to the medium and recording the fluorescence image, the stained medium is photobleached by light irradiation,
A method for detecting a living cell, characterized by recording the fluorescence image and taking a difference image from the image before photobleaching, is disclosed.
However, the gram-negative bacteria in the logarithmic growth phase could not be stained by any of these methods and could not be detected.

[0003] The ability to stain Gram-negative bacteria in the logarithmic growth phase is, for example, the detection of microorganisms such as Escherichia coli contained in foods, the examination of microorganisms in activated sludge in sewage treatment plants, and the examination of rivers and lakes that take up clean water. This means that highly sensitive real-time inspection can be performed in fields where conventional real-time inspection by a staining method was impossible, such as microbial inspection, 24-hour bath microbial inspection, and antibacterial agent quality control.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above-mentioned problems of the prior art. That is, the present invention solves the drawback of the conventional method for detecting living cells, namely, the inability to stain gram-negative bacteria in the logarithmic growth phase, and a method for detecting all living cells including gram-negative bacteria in the logarithmic growth phase. The task to be solved was to be solved.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, added 5-carboxyfluorescein diacetate or 5-sulfofluorescein diacetate to Escherichia coli in a logarithmic growth phase and stained. In this case, it was found that when a calcium salt was added, the bacterium was stained very well and could be detected with high sensitivity, and the present invention was completed.

That is, according to a first aspect of the present invention, there is provided a method for detecting living cells, which comprises dyeing a medium containing cells with a fluorescent enzyme substrate in the presence of calcium ions and measuring the fluorescence. Provided.

According to a second aspect of the present invention, (a) a reagent containing a fluorescent enzyme substrate and a calcium salt, or (b) a combination of a reagent containing a fluorescent enzyme substrate and a reagent containing a calcium salt And a reagent kit for use in the method for detecting a living cell according to the present invention, comprising:

According to a third aspect of the present invention, at least (a) means for holding a medium containing cells, (b) means for adding a fluorescent enzyme substrate and a calcium salt to the medium,
And (c) a device for detecting a live cell, comprising means for measuring the fluorescence intensity of the stained medium.

Preferably, the fluorescent enzyme substrate is a compound that can permeate the cell membrane and can be converted into a fluorescent substance in living cells. Preferably, the fluorescent enzyme substrate is 5-carboxyfluorescein diacetate acetoxymethyl ester, 5- (6-) carboxyfluorescein diacetate, 2 ', 7'-bis- (2-carboxyethyl)-.
5- (6-) carboxyfluorescein acetoxymethyl ester, 5- (6-) sulfofluorescein diacetate, fluorescein diacetate, carsacetacetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5- (6-) carboxyfluoresceindiacetate A compound selected from the group consisting of acetate succinimidyl ester and fluorescein-5-carbonylazide diacetate. Preferably, the live cells are logarithmic growing gram negative bacteria.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method and an embodiment of the present invention will be described in detail. The present invention relates to a method for detecting living cells, which comprises staining a medium containing cells with a fluorescent enzyme substrate and measuring the fluorescence.In the method of the present invention, a medium containing cells in the presence of calcium ions is used. Is stained with a fluorescent enzyme substrate. By performing staining in the presence of calcium ions in this manner, live cells in any medium can be detected with high sensitivity, in real time, easily, and in particular, the logarithm that cannot be detected by the conventional staining method. It has become possible to detect gram-negative bacteria in the growth phase.

As the fluorescent enzyme substrate used in the present invention, a compound which does not emit fluorescence by itself, but which can be converted into a substance which emits fluorescence (fluorescent substance) by the action of an in vivo enzyme such as esterase in living cells Any substance can be used as long as it is a substance that easily permeates the cell membrane. Specific examples of the fluorescent enzyme substrate include, but are not limited to, the following.

5-carboxyfluorescein diacetate acetoxymethyl ester (5-carboxyfluorescein d)
iacetate acetoxymethyl ester; hereinafter, "CFDA-A
M ". ); 5- (6-) carboxyfluorescein diacetate (5- (and-6) carboxyfluore)
scein diacetate; hereinafter may be referred to as "CFDA". ); 2 ', 7'-bis- (2-carboxyethyl) -5- (6-) carboxyfluorescein acetoxymethyl ester (2', 7'-bis- (2-carboxyethyl) -5- (an
d-6) -carboxyfluorescein acetoxymethyl ester; hereinafter may be referred to as "BCECF-AM: 1". ); 5- (6-) sulfofluorescein diacetate;
It may be referred to as “SFDA”. ); Fluorescein diacetate (hereinafter FD)
A ". ); Calcein acetoxymethyl ester;
It may be called "Calcein-AM". ); 5
-Chloromethylfluorescein diacetate (5-chloro
methylfluoresceindiacetate; hereinafter may be referred to as "CMFDA". ); And 5- (6-) carboxyfluorescein diacetate succinimidyl ester (5-
(and 6-) carboxyfluorescein diacetate, succinimidyl
ester; hereinafter may be referred to as “CFDA-SE”. ); And fluorescein-5-carbonylazide diacetat
e; hereinafter, may be referred to as "CFDA azide". ):

These compounds are all known compounds and are commercially available. Of these, CFDA or SFDA is particularly desirable.

The above-mentioned fluorescent enzyme substrate can be used by itself, but can also be used in combination of two or more. Since the fluorescent enzyme substrate is generally poorly soluble in water, when the medium is aqueous, the fluorescent enzyme substrate may be dissolved in a solvent and then added to the medium. Even when the medium is a solid, it may be performed by dissolving the fluorescent enzyme substrate in a solvent and adding the same to the medium. As a solvent for dissolving the fluorescent enzyme substrate, acetone, dimethyl sulfoxide (DMSO), ethanol, water and the like are usually used. It is desirable that these solvents do not deactivate enzymes in living cells to be detected or damage the cells themselves. Moreover, you may melt | dissolve using a nonionic surfactant as needed. After dissolving in such a solvent, the medium is prepared by using water or an appropriate buffer commonly used in biochemistry to an appropriate concentration for staining the medium.

The method of the present invention is characterized in that a medium containing cells is stained in the presence of calcium ions. Calcium ions are usually supplied in the form of a solution containing a calcium salt. The calcium salt used in the present invention may be any one as long as it is water-soluble and produces calcium ions. Specifically, for example,
Calcium chloride, calcium nitrate and the like can be used, and among them, calcium chloride is preferably used.

The calcium salt may be used by dissolving it in water or an appropriate buffer usually used in biochemistry at an appropriate concentration, or may be prepared as a solution together with the fluorescent enzyme substrate. That is, in the method of the present invention, a staining solution containing a fluorescent enzyme substrate and a solution containing a calcium salt may be separately prepared, and they may be separately added to a medium containing cells, or a fluorescent enzyme A solution (hereinafter also referred to as a staining solution) containing a substrate and a calcium salt may be prepared and added to a medium containing cells.

The living cells to be detected by the method of the present invention include:
Microorganisms such as bacteria, yeast, actinomycetes, molds, insect cells such as silkworm Sf9 cells, CHO cells (Proc. Natl. Ac
ad.Sci.USA, 77 , 4216, (1980)), COS-7 cells (A
TCC: CRL1651) and the like, but are not limited thereto, and may be any cells. Gram-negative bacteria in the exponential growth phase are stained only by this method. Examples of the medium in which living cells are to be detected include solid media such as soil, sand, and sampling tape, liquid media such as water, culture solution, and sake, semi-solids such as agar and gel, and mixtures thereof.

The method of adding the fluorescent enzyme substrate and calcium salt (calcium ion) used in the present invention to a medium may be appropriately performed according to the medium. As a method for preparing the medium, when a solid medium such as soil is to be dyed, the fluorescent enzyme is used in the soil as it is, or suspended in water or a suitable buffer commonly used in biochemistry. A staining solution containing a substrate and a calcium salt, or a solution containing a fluorescent enzyme substrate and a solution containing a calcium salt may be added. Depending on the soil, the fluorescent enzyme substrate may be abiotically decomposed by the catalytic action of the soil to increase the background light. In this case, the operation of washing once or twice with water or an appropriate buffer is used. It is effective to add When a solid such as food is used as a medium, a solid that has been ground with a homogenizer or the like and then diluted with water or an appropriate buffer commonly used in biochemistry can be used.

As for the liquid medium, a staining solution containing the above-mentioned fluorescent enzyme substrate and calcium salt, or a solution containing the fluorescent enzyme substrate and a solution containing calcium salt may be added to the liquid medium. When the cell density is low, concentration may be performed using a centrifugation method or the like.

The amount of the fluorescent enzyme substrate to be added to the medium is not particularly limited as long as the medium can be sufficiently stained. Usually, the final concentration in the medium is 1 μM to 1 mM, more preferably SFDA. Is adjusted to 50 μM to 1 mM, and to 1 μM to 0.5 mM for other fluorescent substrates,
It is appropriate to add. If the dye concentration is too low, the absolute value of the fluorescence intensity will decrease, making it unsuitable for detection. Also, even if the dye concentration is too high, the dye becomes insoluble and precipitates, and the background light due to the fluorescent substance outside the cell is strong, and the fluorescence from the cell is hidden, so that the detection efficiency tends to decrease, so it is not suitable. . The mixing ratio between the staining solution and the medium is
There is no particular limitation.

The appropriate amount of the calcium salt to be added to the medium varies depending on the type of the fluorescent enzyme substrate and the cell used. Generally, the higher the concentration of the added calcium salt, the higher the fluorescence intensity of the cell. Tend. Specifically, when an aqueous solution of calcium chloride and SFDA are added to Escherichia coli, the final concentration of the medium is preferably adjusted to about 50 μM to 1 mM.

The order of adding the fluorescent enzyme substrate or calcium salt to the medium is not particularly limited. The calcium salt may be added at an appropriate time before and after the addition of the fluorescent enzyme substrate to the medium, or a solution (preferably an aqueous solution) containing both the fluorescent enzyme substrate and the calcium salt may be added simultaneously to the medium. You may.

The above-mentioned dyeing operation of the medium is carried out in such a manner that the medium is sandwiched between a slide glass and a cover glass, or is placed in a hole slide glass and sealed with a cover glass, or is dyed in an appropriate container such as a plastic container. After performing the operation, the sample is sandwiched between a slide glass and a cover glass and used for measurement of fluorescence.

The method for measuring the fluorescence is not particularly limited. For example, the medium is irradiated with excitation light suitable for light emission induced under a fluorescence microscope or the like, and the intensity of the emitted fluorescence is cooled.
A method of measuring using a detector such as a CD is used. As the excitation light source to be irradiated, a light source generally known as a light source of a fluorescence microscope can be used. Specifically, an ultra-high pressure mercury lamp, an Ar ⁺ laser, or the like can be used. When CFDA is used as a substrate to excite carboxyfluorescein to be produced, A
An r ⁺ laser is preferred. By imaging the thus-detected fluorescence detection values by an image processing system using a microcomputer or the like, specific and real-time detection of living cells is also possible.

The operation procedure in the method for detecting a living cell of the present invention is not particularly limited, and can be performed, for example, as follows. (1) A medium is prepared in a state suitable for adding a staining solution. For example, when the medium is water or the like and the cell density is low, the cells are concentrated using a centrifugal separation method or the like. (2) Dissolve the fluorescent enzyme substrate and calcium salt in an appropriate solvent, and further dissolve in an appropriate buffer to prepare a staining solution. (3) The medium of (1) and the staining solution of (2) are mixed and reacted for an appropriate time to perform staining. (4) The medium of (3) is sandwiched between a slide glass and a cover glass. (5) Irradiate appropriate excitation light under a fluorescence microscope to detect live cells.

The present invention relates to the above-mentioned, which comprises any one of (a) a reagent containing a fluorescent enzyme substrate and a calcium salt, or (b) a combination of a reagent containing a fluorescent enzyme substrate and a reagent containing a calcium salt. The present invention also relates to a reagent kit for use in the method for detecting a living cell according to the present invention. The reagent kit of the present invention is based on the above-described method for detecting a living cell,
It can be prepared using commonly used materials and techniques known per se.

The reagent containing the fluorescent enzyme substrate can be prepared by first dissolving the fluorescent enzyme substrate in a solvent. As a solvent for dissolving the fluorescent enzyme substrate, acetone, dimethyl sulfoxide (DMSO), ethanol, water and the like are usually used. It is desirable that these solvents do not deactivate enzymes in living cells to be detected or damage the cells themselves. Moreover, you may melt | dissolve using a nonionic surfactant as needed. After dissolving in such a solvent, it can be prepared using water or an appropriate buffer usually used in biochemistry to an appropriate concentration for staining the medium. The reagents included in the kit include:
The fluorescent enzyme substrate in a solid state may be used, or the fluorescent enzyme substrate dissolved in a solvent as described above may be used.

The calcium salt-containing reagent can be prepared usually by dissolving it in water or an appropriate buffer commonly used in biochemistry at an appropriate concentration. It may be prepared as a solution together with the fluorescent enzyme substrate described above.

The present invention further provides at least (a) means for holding a medium containing cells, (b) means for adding a fluorescent enzyme substrate and a calcium salt to the medium, and (c) means for adding The present invention also relates to a live cell detection device having means for measuring fluorescence intensity. The live cell detection device of the present invention comprises means sufficient for carrying out the method for detecting a live cell according to the present invention (specifically, at least (a) means for holding a medium containing cells; Any means can be used as long as it has an enzyme substrate and a calcium salt to the medium, and (c) a means for measuring the fluorescence intensity of the stained medium.

Means for holding a medium containing cells include a hole slide glass when the medium is water or a solid suspended in water, and a method for searching for microorganisms attached to the surface of an object. And a slide glass for holding the tape.

The means for adding the fluorescent enzyme substrate and the calcium salt to the medium is not particularly limited as long as the fluorescent enzyme substrate and the calcium salt can be added to the medium held as described above. Although not usually, a container for accommodating a reagent containing a fluorescent enzyme substrate and a calcium salt, or at least a container for accommodating a reagent containing a fluorescent enzyme substrate and a reagent containing a calcium salt separately. It has two containers and a device for dropping liquid from the containers to the holding means.

The means for detecting or measuring the fluorescence or dye in the stained medium includes a fluorescence microscope, and a fluorescence microscope provided with a device for measuring fluorescence intensity or a device for irradiating excitation light is preferred. Further, an image processing system using a microcomputer or the like for imaging the obtained fluorescence detection value may be used. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0033]

EXAMPLES Example 1 (A) Staining of Escherichia coli culture in logarithmic growth phase Escherichia coli (ATCC 11775)
The bacterium was placed in a B broth medium (1% bactotryptone, 0.05% yeast extract, 0.05% sodium chloride), and cultured at 37 ° C. for 1 hour. 300 cultures
After centrifugation at 0 × g for 10 minutes at room temperature, the operation of washing the precipitate with pure water was repeated three times. The precipitates were washed with BTP buffers (20 mM bistrispropane (pH 6.0), 0, 10, 100, 20
(0, 400, 800 mM calcium chloride). As a control, a killed bacterial solution was prepared by fixing the above culture solution with paraformaldehyde (final concentration: 3%) overnight, and a BTP buffer solution having a calcium salt concentration of 800 mM was prepared.
Suspended in 00 μl.

SFDA and CFD as fluorescent enzyme substrates
A was dissolved in dimethyl sulfoxide to a concentration of 50 mM. The bacterial cell fluid and the killed bacterial fluid prepared above were placed in a microtube, and 1 μl of an SFDA solution and a CFDA solution were added thereto, respectively. After standing at room temperature for 90 minutes, centrifugation was performed at 3000 × g for 10 minutes at room temperature, and the precipitate was suspended in pure water. This was suspended in a slide glass having a cylindrical hole of 8 mm in diameter and 1 mm in depth (Ikeda, Japan). (Manufactured by Rika) and sealed with a cover glass (manufactured by Matsunami Glass).

(B) Measurement of Fluorescence and Detection of Live Cells The detection of live cells was performed using an inverted fluorescence microscope (Axiover, manufactured by Zeiss).
t) Below, after counting the number of cells by differential interference, a filter (center wavelength: 490 nm, half width: 10 nm: manufactured by Omega)
Was irradiated with B excitation using an excitation filter, and cells emitting fluorescence were measured. A non-fluorescent fluorescent enzyme substrate is a fluorescent dye (excitation wavelength: 492 nm, fluorescence wavelength: 520 n) due to the activity of esterase contained in all living cells.
m) and observation becomes possible. The result is shown in FIG. It was found that the higher the calcium ion concentration, the higher the proportion of stained cells. In addition, the dead bacteria immobilized with paraformaldehyde were not stained. From the above results, it was revealed that 1) the number of stained cells increases due to the presence of calcium ions, and 2) according to the staining method of the present invention, viable cells of Gram-negative bacteria in the logarithmic growth phase are stained. .

Example 2 Staining of Various Bacteria Cells of various bacteria shown below were cultured at 30 ° C. in an LB broth medium (1% bactotryptone, 0.05% yeast extract, 0.05% sodium chloride). E. coli obtained cells in the logarithmic growth phase cultured for 1 hour and other bacteria for 4 hours, and E. coli cells obtained in the stationary phase cultured for 2 hours and other bacteria for 8 to 15 hours. After the cells were separated by centrifugation, the culture was washed three times with pure water (centrifugation, 10 minutes, 3, 0
(00 g, room temperature). 100 μl of 20
mM BTP buffer (final calcium ion concentration: 40
(0 mM) and 1 μl of a 50 mM SFDA solution and 5 μl of the bacterial solution were added. After 90 minutes, the cells were observed with a fluorescence microscope. As a result, in all strains, cells in both the logarithmic growth phase and stationary phase were clearly stained. From these results, it was shown that this staining method is effective for detecting many types of cells.

Bacteria used in Example 2 (Gram-negative bacteria) Acinetobacter lwoffii (ATCC: 15309) Agrobacterium radiobacter (IAM: 12048) Alcaligenes xylosoxydans subsp.xylosoxydans (IAM: 1
2684) Brevundimonas diminuta (JCM: 2788) Comamonas testosteroni (ATCC: 11996) Escherichila coli (ATCC: 11775) Pseudomonas putida (ATCC: 12633) Serratia marcescens (IFO: 12648)

(Gram-positive bacteria) Arthrobacter globiformis (IFO: 12137) Corynebacterium glutamium (ATCC: 13032) Rhodococcus rhodochrous (ATCC: 13808) Bacillus subtilis (ATCC: 6051) Micrococcus luteus (ATCC: 9341) Staphylococcus aureus (ATCC: 12600)

[0039]

According to the method of the present invention, living cells in any medium can be detected with high sensitivity and in a simple, real-time manner. In particular, since the Gram-negative bacteria in the logarithmic growth phase, which cannot be detected by the conventional staining method, are stained, the method of the present invention is particularly effective for samples such as foods.

[Brief description of the drawings]

FIG. 1 is a diagram showing the calcium ion concentration and the number of stained cells at the time of E. coli staining. In the figure, the hatched and white graphs indicate staining with SFDA and CFDA, respectively, and the results of staining dead cells as a control. The staining experiment with SFDA was performed three times, and the graph shows the average and the standard deviation.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/48 G01N 33/48 P // (C12Q 1/06 (C12Q 1/06 C12R 1:01) C12R 1:01) (72) Inventor Yoshitaka Yoshimura 11 Minamiotani, Machida-shi, Tokyo Inside the Mitsubishi Chemical Life Science Research Institute, Inc. (72) Inventor Kawasaki Yukishige 11 Minamiotani, Machida-shi, Tokyo Mitsubishi Chemical Life Co., Ltd. Inside the Science Research Institute (72) Inventor Takashi Tsuji 11 Minamiotani, Machida-shi, Tokyo Inside Mitsubishi Chemical Life Science Institute (72) Inventor Ryuichiro Kurane 1-3-3 Higashi 1-3-3 Tsukuba, Ibaraki Pref. Ministry of International Trade and Industry F-term (Reference) 2G043 AA03 BA17 DA02 EA01 FA02 KA09 LA03 2G045 AA28 BB10 BB25 CB21 FA12 FA16 FB01 FB12 GC22 2G054 AA10 AB10 CA30 CE10 EA03 GB10 4B029 AA07 BB01 BB02 FA01 FA09 4B063 QA01 QQ05 QQ06 QQ08 QQ15 QQ16 QQ18 QQ19 QR50 QR58 QS36 QS39 QX02

Claims (9)

[Claims] 1. A method for detecting living cells, comprising staining a medium containing cells with a fluorescent enzyme substrate in the presence of calcium ions and measuring the fluorescence. 2. The method according to claim 1, wherein the fluorescent enzyme substrate is capable of permeating a cell membrane,
The method according to claim 1, wherein the compound is a compound capable of changing into a fluorescent substance in living cells. 3. The method according to claim 1, wherein the fluorescent enzyme substrate is 5-carboxyfluorescein diacetate acetoxymethyl ester,
-(6-) carboxyfluorescein diacetate,
2 ', 7'-bis- (2-carboxyethyl) -5
(6-) carboxyfluorescein acetoxymethyl ester, 5- (6-) sulfofluorescein diacetate, fluorescein diacetate, carsacetacetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5- (6-) carboxyfluorescein diacetate succinate The method according to claim 1 or 2, which is a compound selected from the group consisting of sinimidyl ester and fluorescein-5-carbonylazide diacetate. 4. The method according to claim 1, wherein the living cells are gram-negative bacteria in the logarithmic growth phase. 5. The method according to claim 1, comprising: (a) a reagent containing a fluorescent enzyme substrate and a calcium salt; or (b) a combination of a reagent containing a fluorescent enzyme substrate and a reagent containing a calcium salt. 5. A reagent kit for use in the method for detecting a living cell according to any one of items 1 to 4. 6. The fluorescent enzyme substrate is capable of permeating a cell membrane,
The reagent kit according to claim 5, which is a compound capable of changing into a fluorescent substance in living cells. 7. The method according to claim 7, wherein the fluorescent enzyme substrate is 5-carboxyfluorescein diacetate acetoxymethyl ester,
-(6-) carboxyfluorescein diacetate,
2 ', 7'-bis- (2-carboxyethyl) -5
(6-) carboxyfluorescein acetoxymethyl ester, 5- (6-) sulfofluorescein diacetate, fluorescein diacetate, carsacetacetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5- (6-) carboxyfluorescein diacetate succinate The reagent kit according to claim 5, which is a compound selected from the group consisting of sinimidyl ester and fluorescein-5-carbonylazide diacetate. 8. The reagent kit according to claim 5, wherein the living cells are gram-negative bacteria in the logarithmic growth phase. 9. At least (a) a means for holding a medium containing cells, (b) a means for adding a fluorescent enzyme substrate and a calcium salt to the medium, and (c) a fluorescent intensity of the stained medium. A live cell detection device comprising means for measuring.