JP4286531B2 - Bacteria Gram stain discrimination method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for discriminating the gram stainability of microorganisms with a simple operation.
[0002]
[Prior art]
When handling a microorganism, it is necessary to identify the genus and species of the microorganism, and the first step in identifying the microorganism is a test on the gram stainability of the microorganism. The identification operation to be performed next for this microorganism varies depending on the result of the Gram stainability.
[0003]
Therefore, the Gram stain discrimination method, which is the first identification step for bacteria, needs to be quick and accurate.
This method of distinguishing Gram stain of bacteria was found by Christian Gram, and this Gram stain distinguishing method has been improved since then, but both methods are heat-fixed smear cells. By staining the sample with basic crystal purple and then with a dilute iodine solution, and then treating the specimen with an organic solvent for a short time, the Gram-positive cells are resistant to decolorization and remain stained in a deep blue-purple color. Gram-negative cells are rapidly and completely decolored and stained with a third solution (fuchsin, safranin, etc.), so that both are discriminated by blue-violet or blue. In this conventional method for distinguishing Gram stain, it is essential to use proliferating cells.
[0004]
As described above, the conventional method has a problem that it takes a great number of steps for discrimination and requires skill to perform each step.
Therefore, it has been desired to develop a simpler method for identifying the gram stainability of bacteria.
[0005]
OBJECT OF THE INVENTION
An object of the present invention is to provide a method capable of easily identifying the gram stainability of bacteria.
[0006]
Summary of the Invention
According to the method for distinguishing Gram stain of bacteria of the present invention, an aqueous solution of an alkali metal hydroxide is at least one basic substance selected from the group consisting of crystal violet, basic fuchsin, gentian violet B, and gentian violet R. Bacteria are added to and mixed with the discrimination solution to which the dye solution has been added, and Gram-negative bacteria and Gram-positive bacteria are differentiated by fading of the mixture.
In the discrimination method of the present invention, it is preferable to use potassium hydroxide and / or sodium hydroxide as the alkali metal hydroxide.
[0008]
Furthermore, in the present invention, the pH value of the discrimination liquid is preferably adjusted to 12 or more.
In the present invention, the concentration of the basic dye in such a basic dye solution is preferably adjusted within the range of 0.01 to 0.2% by weight.
The gram-staining property of the bacterium of the present invention is discriminated by using the difference in fading between gram-positive and gram-negative bacteria with respect to the basic dye using an aqueous solution of a basic dye and an aqueous alkali metal hydroxide. It does not require special equipment such as a microscope and is extremely easy to operate. Although the operation is simple and does not require a specific device, the accuracy of discriminating between gram-positive bacteria and gram-negative bacteria is high.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Next, the Gram staining determination method of the present invention will be specifically described.
The Gram stain determination method of the present invention is to determine the Gram stain property of bacteria by a simple method, and uses an aqueous solution of an alkali metal hydroxide and an aqueous solution of a basic dye.
[0010]
The alkali metal hydroxide used in the method of the present invention is a metal hydroxide of Group IA of the periodic table, and specifically includes lithium hydroxide, sodium hydroxide, potassium hydroxide and the like. Can do. Among these, in the present invention, sodium hydroxide and potassium hydroxide can be used alone or in combination. When this sodium hydroxide and potassium hydroxide are compared, there is a tendency that Gram dyeability is easily discriminated when potassium hydroxide is used.
[0011]
The alkali metal hydroxide as described above is dissolved in water and used as an aqueous solution. In the present invention, the concentration of the alkali metal hydroxide in the aqueous solution is usually 0.1 to 10% by weight / volume%, preferably 1 to 5% weight / volume%, particularly preferably 2 to 4% weight / volume%. It is adjusted to become.
Separately, an aqueous solution of a basic dye is prepared. The basic dye that can be used in the present invention is any one of gentian violet, basic fuchsin, auramin, crystal violet, safranine, toluidine blue, neutral red, bismarck brown, methylene blue, methylene violet and rhodamine. Is preferably used.
[0012]
In the present invention, it is particularly preferable to use any one of gentian violet, basic fuchsin, and crystal violet. The gentian violet includes gentian violet R and gentian violet B, and any of these can be used in the present invention.
These basic dyes are dissolved in water and used as an aqueous solution. In the present invention, the concentration of the basic dye in the aqueous solution is usually 0.001-1.000 wt / vol%, preferably 0.005-0.500 wt / vol%, particularly preferably 0.010- It is prepared to be in the range of 0.150% weight / volume. The concentration of this basic dye in the aqueous solution is determined by adding this basic dye aqueous solution to the above-mentioned alkali metal hydroxide aqueous solution. The amount can be reduced, or the concentration can be lowered to increase the amount added.
[0013]
The discriminating method of the present invention is a method for discriminating the Gram stainability of bacteria using an aqueous solution of an alkali metal hydroxide prepared as described above and an aqueous solution of a basic dye.
That is, in the method of the present invention, for example, when a test tube having a length of 100 mm and a diameter of 10 mm (internal volume; approximately 8.5 ml) is used, the alkali metal hydroxide aqueous solution prepared as described above is reduced to 0. Collect 1 to 1 ml, preferably 0.3 to 0.5 ml in multiple containers. Here, the amount of the aqueous solution with respect to the container is at least an amount capable of distinguishing the color. For example, see the relationship between the liquid height (liquid amount) in the test tube and the amount of the alkali metal hydroxide aqueous solution to be added. Thus, the liquid volume can be adjusted in accordance with the volume of the container.
[0014]
Next, an aqueous solution of the basic dye prepared as described above is added to each container and rapidly stirred to prepare a discrimination liquid. At this time, the addition amount of the aqueous solution of the basic dye is about one drop in each container, and this amount is 0.001 to 0.001 to 1 mg of the alkali metal hydroxide in the aqueous solution collected in the container. It corresponds to 0.005 mg, preferably 0.002 to 0.003 mg. That is, 1 ml of the basic dye aqueous solution is about 30 drops in terms of the number of water drops. Therefore, one drop of the basic dye aqueous solution is usually about 0.03 ml, for example, in a solution of 0.08 g / 100 ml. About 1 to 30 μg of basic dye is contained in one drop of the solution collected from 1).
[0015]
The discriminating solution thus prepared usually shows alkalinity, but in the discriminating method of the present invention, the determination accuracy is improved by setting the pH value of the discriminating solution to 12 or more, preferably 13 or more. By adding a buffer solution, the pH value of this discrimination solution can be prepared as described above.
An appropriate amount of the bacteria to be identified is caught with a platinum loop or the like, and is put into the discrimination solution prepared as described above and mixed quickly.
[0016]
The bacteria to be discriminated here are fished so that the amount of the basic dye is usually in the range of ⁵ to 50 μg, preferably 9 to 30 μg per 10 ⁵ to 10 ⁶ bacteria. (0.33 ml) is usually fished so that 10 ⁴ to 10 ⁷ bacteria, preferably 10 ⁵ to 10 ⁶ bacteria are present.
In the discrimination method of the present invention, the color fading speed of the mixed liquid (discrimination liquid in which bacteria are suspended) prepared as described above is compared with the fading speed of the discrimination liquid not added with bacteria (blank). , Those with a fading speed faster than the blank (bacteria whose liquid color disappears) are Gram-positive bacteria, and those with the same or slower color fading speed as the blank (bacteria whose liquid color does not disappear) are Gram-negative bacteria. is there. That is, the color of the discrimination liquid in which an aqueous solution of a basic dye is dropped into an aqueous solution of an alkali metal hydroxide is not changed at the beginning of preparation. However, when a Gram-positive bacterium is contacted, this discrimination liquid rapidly disappears ( This fading speed is no longer than the fading speed of the discriminating liquid itself (the discriminating liquid that does not contact anything). This discriminating solution itself also fades (decolors) with the passage of time, but normally it takes more time for fading than when Gram-positive bacteria are contacted. On the other hand, when Gram-negative bacteria are in contact, the color fading speed of the discrimination liquid is significantly slower than the fading speed of the discrimination liquid itself. The discriminating solution used in the present invention fades with time as described above, and this fading rate is not so different from the fading rate when gram-positive bacteria are contacted. The color fading when the positive bacteria are contacted may be almost the same time or the color fading of the blank may be faster than the discrimination liquid that has contacted the gram positive bacteria. However, even in such a case, since there is a significant difference from the color fading state of the discrimination liquid that has come into contact with the Gram-negative bacteria, it is hardly possible to make a discrimination mistake. Also, if the discrimination is subtle in this way, the fading state of the bacteria trying to discriminate Gram staining by simultaneously contacting the bacteria to be identified with standard Gram-negative and Gram-positive bacteria. Compared with the color fading state of these standard positive bacteria and negative bacteria, the accuracy of the discrimination becomes remarkably high.
[0017]
That is, according to the method of the present invention, Gram-positive bacteria and Gram-negative bacteria can be differentiated from the fading difference from the blank, but in addition to the bacteria and blanks to be distinguished, A suitable amount of gram-positive bacteria (for example, Staphylococcus aureus) and Gram-negative bacteria (for example, Escherichia coli, Pseudomonas aeruginosa), etc. at the same time in different containers, By comparing the discoloration rate of the bacteria to be discriminated, it is possible to more accurately determine the Gram stainability of the bacteria.
[0018]
As described above, the alkali metal hydroxide aqueous solution and the basic dye aqueous solution used in the method of the present invention change over time due to oxygen in the air and so on. It is desirable to do.
In addition, a discrimination liquid in which an aqueous solution of a basic dye is added to an aqueous solution of an alkali metal hydroxide fades with time, and the method of the present invention fades after adding an aqueous solution of a basic dye. The determination of the Gram staining property is based on the property that the property is promoted or delayed by Gram-positive or Gram-negative bacteria. Ideally, it is performed so that at least the preparation step and the bacterium contact step do not cause a time difference more than necessary. Further, when a time difference occurs due to the test, it is necessary to consider the generated time difference in the determination.
[0019]
As described above, according to the present invention, it is possible to accurately and accurately discriminate the gram stainability of bacteria performed at the first stage of bacterial identification.
[0020]
【The invention's effect】
According to the present invention, it is possible to distinguish bacteria as Gram-positive bacteria and Gram-negative bacteria with a very simple operation. Therefore, by adopting the method of the present invention in the first stage for identifying unknown bacterial genera and species, bacterial identification can be performed efficiently.
[0021]
That is, the method of the present invention does not stain bacteria with a dye, but utilizes the characteristic of bacteria that the discoloration state of the basic dye solution is changed by the bacteria. The operation of fixing is unnecessary, and the Gram stainability of bacteria can be determined in a short time. Therefore, according to the present invention, the bacteria Gram stainability is discriminated from the contrast with the fading time of the dye in the blank prepared at the same time. Therefore, no expensive device or complicated device is required, and the bacteria Gram stainability is visually observed. Can be accurately determined in a short time, and no particular skill is required for this determination.
[0022]
【Example】
Next, the method for discriminating the gram stainability of bacteria according to the present invention will be described with reference to examples, but the present invention is not limited thereto.
[0023]
[Example 1]
3 g of potassium hydroxide (KOH) was weighed and placed in a 100 ml volumetric flask, and distilled water was added to prepare 100 ml of a 3 wt / vol% potassium hydroxide solution. Separately, 0.08 g of crystal violet B was weighed and placed in a 100 ml volumetric flask, and distilled water was added to make 100 ml in the same manner as above to prepare a 0.08 weight / volume% crystal violet solution.
[0024]
Place 0.3 ml of the potassium hydroxide aqueous solution prepared as described above in three test tubes, then take the crystal violet solution into a dropper, drop it into each test tube and shake quickly. The discrimination liquid was prepared by homogenization. The pH value of this discrimination solution was 14. The crystal violet contained in one drop of the solution is about 24 μg.
[0025]
E. coli (E. coli, Gram-negative bacteria) is crammed into one of the above test tubes, and S. aureus (Gram-positive bacteria) is platinum-eared into the other test tube. Then, these test tubes were shaken without putting anything in the last test tube, and the color fading state of the discrimination liquid was observed. The number of added bacteria is about 10 ⁵ .
[0026]
By operating as described above, the color of the liquid in which Staphylococcus aureus that is a Gram-positive bacterium was added disappeared in about 5 seconds, and then the color of the discrimination liquid in the test tube in which nothing was added as a blank disappeared (approximately 10 seconds later), and about 15 seconds later, the color of the discrimination solution into which E. coli, which is a Gram-negative bacterium, was added disappeared.
As described above, gram-negative bacteria and gram-positive bacteria were clearly different in the color fading state of the discrimination solution, and the gram staining ability of the bacteria could be distinguished easily and reliably.
[0027]
[Example 2]
In Example 1, the gram stainability of the bacteria was examined in the same manner except that sodium hydroxide was used instead of potassium hydroxide.
As a result, the color of the liquid in which Staphylococcus aureus that is a Gram-positive bacterium was added in about 5 seconds disappeared, and then the color of the discrimination liquid in the test tube to which nothing was added as a blank disappeared (after about 10 seconds). After about 15 seconds, the color of the discrimination solution into which Escherichia coli, which is a Gram-negative bacterium, was added disappeared.
[0028]
As described above, gram-negative bacteria and gram-positive bacteria were clearly different in the color fading state of the discrimination solution, and the gram staining ability of the bacteria could be distinguished easily and reliably.
[0029]
[Example 3]
In Example 1, the gram stainability of the bacteria was examined in the same manner except that lithium hydroxide was used instead of potassium hydroxide.
As a result, the color of the discriminating solution in the test tube in which nothing was added as a blank in about 10 seconds disappeared, and after several seconds, the discriminating solution into which Staphylococcus aureus as a gram-positive bacterium was added, and Escherichia coli as a gram-negative bacterium were introduced. The liquid color disappeared in the order of the discrimination liquid.
[0030]
Although it was possible to discriminate between gram-negative and gram-positive bacteria by using lithium hydroxide as described above, the difference in fading time is small, and it is harder to discriminate than when potassium hydroxide is used. all right.
[0031]
Examples 4 to 6
In Example 1, basic fuchsin was used instead of crystal violet, and potassium hydroxide aqueous solution (Example 4), sodium hydroxide (Example 5), and lithium hydroxide (Example 6) were used independently. Except for the above, the Gram stainability of the bacteria was examined in the same manner.
[0032]
As a result, when any of potassium hydroxide aqueous solution (Example 4), sodium hydroxide (Example 5), and lithium hydroxide (Example 6) is used, yellow grapes that are Gram-positive bacteria in about 5 seconds. The color of the solution containing the cocci disappeared, then the color of the discriminating solution in the test tube to which nothing was added as a blank disappeared (after about 10 seconds), and further, Escherichia coli, which is a Gram-negative bacterium, was introduced after about 15 seconds. The color of the discrimination liquid disappeared.
[0033]
By using basic fuchsin as described above, there is no difference in fading time regardless of whether potassium hydroxide, sodium hydroxide, or lithium hydroxide is used, and gram-negative and gram-positive bacteria can be distinguished. did it.
[0034]
Examples 7 to 8
Gram staining of bacteria in the same manner as in Example 1, except that gentian violet B was used instead of crystal violet, and an aqueous potassium hydroxide solution (Example 7) and sodium hydroxide (Example 8) were used independently. I examined the sex.
As a result, when using either an aqueous potassium hydroxide solution (Example 7) or sodium hydroxide (Example 8), the color of the solution into which Staphylococcus aureus that is a Gram-positive bacterium was added disappeared in about 5 seconds. Next, the color of the discriminating solution in the test tube to which nothing was added as a blank disappeared (after about 10 seconds), and further, the color of the discriminating solution into which Escherichia coli as a gram-negative bacterium was added disappeared after about 15 seconds. The time difference between potassium hydroxide and sodium hydroxide was relatively large.
[0035]
[Example 9]
In Example 1, gentian violet B was used in place of crystal violet, and the bacterial Gram stainability was examined in the same manner except that lithium hydroxide was used.
As a result, the color of the discriminating solution in the test tube in which nothing was added as a blank in about 10 seconds disappeared, and after several seconds, the discriminating solution into which Staphylococcus aureus as a gram-positive bacterium was added, and Escherichia coli as a gram-negative bacterium were introduced. The liquid color disappeared in the order of the discrimination liquid.
[0036]
Although it was possible to discriminate between gram-negative and gram-positive bacteria by using lithium hydroxide as described above, the difference in fading time is small, and it is harder to discriminate than when potassium hydroxide is used. all right.
[0037]
Examples 10-12
In Example 1, instead of crystal violet, Gentian Violet R was used, and potassium hydroxide aqueous solution (Example 10), sodium hydroxide (Example 11), and lithium hydroxide (Example 12) were used independently. Except for the above, the Gram stainability of the bacteria was examined in the same manner.
[0038]
As a result, when any of potassium hydroxide aqueous solution (Example 10), sodium hydroxide (Example 11), and lithium hydroxide (Example 12) is used, yellow grapes that are Gram-positive bacteria in about 5 seconds. The color of the solution in which the cocci were added disappeared, then the color of the discriminating solution in the test tube that had not been added as a blank disappeared (approximately 10 seconds later), and after approximately 15 seconds, E. coli, which was a gram-negative bacterium, was introduced. Although the liquid color of the discrimination liquid disappeared, the time difference when lithium hydroxide was used was the largest, and discrimination was easier compared with the case where potassium hydroxide and sodium hydroxide were used.
[0039]
[Comparative Examples 1-3]
In Example 1, edible yellow No. 5 (Comparative Example 1), edible red No. 3 (Comparative Example 2), and edible green No. 3 (Comparative Example 3) were used in place of Crystal Violet, respectively. An attempt was made to examine the Gram stainability of bacteria in the same manner except that sodium oxide and lithium hydroxide were used, but no discrimination was made.
[0040]
Examples 13 to 16
In Example 10, the concentration of gentian violet R was 0.03 weight / volume% (Example 13), 0.05 weight / volume% (Example 14), 0.08 weight / volume% (Example 15), Gram stainability of the bacteria was determined in the same manner except that the content was changed to 0.10 weight / volume% (Example 16).
[0041]
As a result, it was possible to discriminate between Gram-negative bacteria and Gram-positive bacteria with any discriminating solution, but 0.08 weight / volume% (Example 15) was the easiest to distinguish, and 0.03 weight / volume. % (Example 13) and 0.10% weight / volume (Example 16), the time difference was small and it was difficult to distinguish.
[0042]
[Example 17]
In Example 10, when the buffer solution was added and the pH value of the discrimination solution was 12.5, 13.0, 13.5, 14.0, 14.0 or more, respectively, the Gram staining property of the bacteria was discriminated. In any discriminant, it was possible to discriminate between Gram-negative bacteria and Gram-positive bacteria, but it was most easily discriminated when the pH value was 14.0 or higher, and the discriminant having a pH value of 12.5 was used. In such a case, it is most difficult to discriminate and it is easy to discriminate as a whole at pH values of 13.0, 13.5, and 14.0, but there is a tendency that discrimination becomes easier as the value increases.
[0043]
Example 18
In Example 10, the Gram staining test was performed in the same manner except that Bacilus was used as the Gram-positive bacterium, Burkholderia cepacia as the Gram-negative bacterium, and Serratia liquefaiens as the Gram-negative bacterium. In addition, it was possible to distinguish between gram-negative and gram-positive bacteria.
[0044]
As described above, according to the gram stainability determination method for bacteria of the present invention, the gram stainability of bacteria can be determined by a very simple operation. Moreover, the discrimination method of the present invention does not stain bacteria, but visually discriminates the difference in the fading time of the discrimination liquid due to bacteria, and does not require a special device. In addition, the operation is simple, and no skill is required for carrying out the discrimination method of the present invention.
[0045]
Such a discrimination method of the present invention can be applied to almost all bacteria, and although there is some variation in the sharpness of discrimination, as long as it is confirmed, the obtained result is consistent with the Gram staining property of the bacteria used. did.

Claims (4)

Bacteria is added to a discrimination liquid in which at least one basic dye solution selected from the group consisting of crystal violet, basic fuchsin, gentian violet B, and gentian violet R is added to an aqueous solution of an alkali metal hydroxide. A method for distinguishing Gram-staining of bacteria, comprising mixing and distinguishing Gram-negative bacteria from Gram-positive bacteria by fading the mixture.   2. The bacteria Gram-staining determination method according to claim 1, wherein the alkali metal hydroxide is potassium hydroxide and / or sodium hydroxide.   2. The method for discriminating bacterial Gram staining according to claim 1, wherein the pH value of the discrimination liquid is adjusted to 12 or more.   2. The Gram stain determination method according to claim 1, wherein the concentration of the basic dye in the basic dye solution is in the range of 0.01 to 0.2% by weight.