JP2017099328A - Post-dyeing reagent for gram dyeing and gram dyeing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new Gram dyeing method that enhances the post-dyeing ability of fuchsin and can favorably dye bacteria such as Helicobacter spp.SOLUTION: A post-dyeing reagent for Gram dyeing comprises (A) basic fuchsin, (B) a lower alcohol, and (C) a base for regulating pH in 4.5-7.5.SELECTED DRAWING: None

Description

  The present invention relates to a Gram dyeing method and a post-staining reagent used in this method.

Gram staining is one of the methods of dyeing bacteria with pigments and is an important staining method that is the basis of bacterial taxonomy. Bacteria can be roughly classified into two types (gram positive and gram negative) by Gram staining.
The difference in staining is due to the difference in the structure of the bacterial wall. The large difference in cell wall seen between Gram-positive and Gram-negative bacteria reflects the significant biological difference between the two, and Gram staining is an important technique for bacterial classification.

For Gram staining, the sample is treated with crystal violet and stained (pre-stain treatment: Gram positive and gram negative are stained purple at this stage), then treated with iodine (mordanting treatment), and then decolorized with an ethanol solution. Finally, a method (Hucker's modified method) of treating with a safranine solution or fuchsin solution (post-staining treatment) is widely known. In this method, Gram-positive bacteria are stained purple, and Gram-negative bacteria are stained red. In this method, anaerobic bacteria, Campylobacter spp. Helicobacter spp., Etc. have weak staining and are difficult to judge.
As a means of improving the staining method for these difficult-to-stain bacteria, a method of adding 5% sodium bicarbonate after crystal violet treatment has been developed (Burmy's modified method). However, in this method, the dyeing step is increased. Sodium bicarbonate is less effective after long-term storage, so it must be prepared frequently. The mixture of staining solution and sodium bicarbonate undergoes a chemical change in a short period of time. This causes problems such as inability to mix.

  On the other hand, a method of using Victoria Blue as the first dye for dyeing and a picric acid-ethanol solution as a mordant reagent (Nishioka method) has also been developed. However, this method has the problem that Helicobacter spp. And the like have poor dyeability and post-dyeing dyeability is weak.

  Furthermore, a method of adding an anionic surfactant to the sample, adding methylene blue and then extracting with chloroform, and observing the presence or absence of coloring of the chloroform phase (Patent Document 1), crystal violet and anionic surfactant as pre-staining reagent A method of using an agent (Patent Document 2) has been reported.

JP 7-313192 A JP 2003-169694 A

However, even the methods of Patent Documents 1 and 2 have not solved the problem that it is difficult to determine some bacteria such as Helicobacter spp.
Therefore, an object of the present invention is to provide a new Gram staining method that enhances the post-staining property with fuchsin and can stain well bacteria such as Helicobacter spp.

Therefore, the present inventor has made various studies on post-staining treatment, which has heretofore been garnering little attention. As a post-staining test solution, the pH is adjusted to 4.5 to 7.5 in addition to basic fuchsin and lower alcohol It was found that if a test solution containing a base was used, the post-staining property was remarkably improved and bacteria such as Helicobacter spp. Could be stained well. Moreover, when a chelating agent was added to this, it discovered that the long-term storage stability of a post dyeing test liquid also improved, and completed this invention.

  That is, the present invention provides the following [1] to [6].

[1] A post-dyeing test solution for Gram dyeing containing (A) basic fuchsin, (B) a lower alcohol, and (C) a base having a pH of 4.5 to 7.5.
[2] The post-dyeing test solution for Gram dyeing according to [1], further comprising (D) a chelating agent.
[3] The post-dyeing test solution for Gram dyeing according to [1] or [2], wherein the component (A) is basic fuchsin.
[4] The post-dye for Gram dyeing according to [2] or [3], wherein (D) the chelating agent is a chelating agent selected from aminopolycarboxylic acid, aromatic or aliphatic carboxylic acid, phosphonic acid and hydroxycarboxylic acid Test solution.
[5] (1) A step of treating a sample with a pre-staining reagent containing crystal violet or Victoria blue, (2) A step of treating with a mordanting reagent containing iodine or picric acid, (3) containing a polar organic solvent A gram dyeing method comprising a step of treating with a decolorizing reagent and a step of treating with a post-gram dyeing reagent for gram dyeing according to any one of (4) [1] to [4].
[6] The Gram dyeing method according to [5], comprising a step of treating with an aqueous carbonate solution or an aqueous hydrogen carbonate solution between step (1) and step (2).

If the post-staining reagent of the present invention is used, Helicobacter has been difficult to detect due to its weak staining ability.
Since bacteria such as spp. can be clearly stained, it is possible to separate a wide range of Gram-positive and Gram-negative bacteria. Further, the post-staining agent of the present invention containing a chelating agent does not cause precipitation even after long-term storage and is particularly useful as a kit reagent.

The dyeing | staining result by Example 1 is shown. The dyeing | staining result by the comparative example 1 is shown.

  The post-dyeing reagent for Gram dyeing of the present invention contains (A) basic fuchsin, (B) a lower alcohol, and (C) a base that adjusts the pH to 4.5 to 7.5.

  (A) Basic fuchsin is a dye for staining cell nuclei. Basic fuchsin is a staining pigment containing fuchsin, and usually a mixture of rose aniline, rose rose aniline, new fuchsin, magenta 2, and the like is used. Moreover, the Peyfer liquid which is a dilution liquid of a carboxylic acid fuchsin liquid can also be used.

  The basic fuchsin concentration in the post-dying test solution is preferably 0.005 to 0.2% by mass, more preferably 0.005 to 0.1% by mass, and 0.02 to 0.04% by mass from the viewpoint of dyeability. Is more preferable.

  (B) As a lower alcohol, C1-C5 alcohols, such as methanol, ethanol, propanol, isopropanol, are mentioned, However, Ethanol is more preferable. The lower alcohol is used to enhance dyeability, and the concentration in the post-dyeing test solution is preferably 1 to 40% by mass, more preferably 5 to 30% by mass, from the viewpoint of dyeability. More preferred is mass%.

  (C) The base that adjusts the pH to 4.5 to 7.5 is a base that adjusts the pH of the post-staining test solution to 4.5 to 7.5. When the pH of the post-staining test solution is less than 4.5, the dyeability is not sufficient. For example, the pH of the basic fuchsin and lower alcohol-containing aqueous solution is about 4.2, and the dyeability is not sufficient. On the other hand, if the pH of the post-staining test solution exceeds 7.5, the stability of the post-staining test solution is not sufficient and precipitation occurs. More preferably, the pH of the buffer is 4.5 to 7.5, more preferably 5.0 to 7.3, and still more preferably 6.0 to 7.0.

  The base for adjusting the pH to 4.5 to 7.5 may be an alkali such as sodium hydroxide, but a buffering agent for adjusting the pH to 4.5 to 7.5 is preferable from the viewpoint of stabilizing the pH. Examples of the buffer include Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), 2-morpholinoethanesulfonic acid (MES), N, N-bis (2-hydroxyethyl) -2-amino. Ethanesulfonic acid (BES), tris (hydroxymethyl) aminomethane (Tris), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), 3-morpholinopropanesulfonic acid (MOPS), N-tris ( Good buffer such as hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), phosphate buffered saline (PBS), etc. Among them, Bis-Tris, MES, BES, HEPES, PBS, etc. are preferred. Arbitrariness. These can use 1 type (s) or 2 or more types.

The post-dyeing test solution of the present invention can contain (D) a chelating agent for the purpose of further improving long-term storage stability. Addition of a chelating agent does not cause precipitation for a long period of 4 months or longer.
As the chelating agent, any compound having the ability to chelate metal ions may be used. For example, aminopolycarboxylic acid, aromatic or aliphatic carboxylic acid, amino acid, ether polycarboxylic acid, phosphonic acid, hydroxycarboxylic acid, phosphoric acid series Etc. Of these, aminopolycarboxylic acids, aromatic or aliphatic carboxylic acids, phosphonic acids, and hydroxycarboxylic acids are more preferred.

As aminopolycarboxylic acid, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), N- (2-hydroxyethyl) iminodiacetic acid (HIMDA), diethylenetriaminepenta Examples include acetic acid (DTPA), N- (2-hydroxyethyl) ethylenediaminetriacetic acid (EDTA-OH), and glycol ether diamine tetraacetic acid (GEDTA) or salts thereof.
Aromatic or aliphatic carboxylic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, itaconic acid, aconitic acid, pyruvic acid, salicylic acid, acetylsalicylic acid, hydroxybenzoic acid, aminobenzoic acid, phthalic acid, Trimellitic acid, gallic acid, and salts thereof.
Examples of the phosphonic acid include imidinophosphonic acid, alkyl diphosphonic acid, 1-hydroxyethane-1,1-diphosphonic acid, and salts thereof. Examples of the hydroxycarboxylic acid include citric acid, gluconic acid, tartaric acid, and salts thereof.

  The concentration of the chelating agent in the post-staining reagent is preferably from 0.01 mM to 100 mM, more preferably from 0.05 mM to 10 mM, and even more preferably from 0.1 mM to 1 mM, from the viewpoint of long-term storage stability.

  The post-staining test solution may contain water in addition to the above components.

By using the post-staining reagent of the present invention, bacteria such as Helicobacter spp., Which have been poor in dyeability, can be dyed well. The Gram staining method using the post-staining test solution of the present invention includes (1) a step of treating a sample with a pre-staining test solution containing crystal violet or Victoria blue, and (2) a treatment with a mordanting test solution containing iodine or picric acid. Gram dyeing method characterized by including the process, (3) The process processed with the decoloring test liquid containing a polar organic solvent, and (4) The process processed with the post-dying test liquid of the said this invention is mentioned.
Hereinafter, each step (1) to (4) will be described.

  The sample used in step (1) is a sample containing test bacteria. The sample is, for example, a smear prepared by thinly and uniformly smearing clinical specimens or isolates on a slide glass, naturally drying, and then flame-fixing or methanol-fixing.

  As the pre-staining solution containing crystal violet or Victoria blue, an aqueous solution containing crystal violet or Victoria blue is preferable. Crystal violet is preferably contained in an amount of 0.05 to 2% by mass, more preferably 0.2 to 0.6% by mass in the pre-staining test solution. Moreover, it is preferable to contain 0.1 to 0.2 mass% of Victoria blue in the pre-dying test solution.

  The pre-staining test solution may further contain an anionic surfactant, a lower alcohol, a buffering agent and the like. Examples of the anionic surfactant include amino acid anionic surfactants, alkylbenzene sulfonates, and alkyl sulfates. Examples of the lower alcohol include ethanol.

  The sample may be treated with the pre-staining reagent, for example, by adding a pre-treatment reagent to the sample and reacting at room temperature for 30 seconds to 1 minute. After treatment, the sample is washed with water.

After the step (1) is completed, the sample is preferably treated with an aqueous carbonate solution or an aqueous hydrogen carbonate solution from the viewpoint of improving dyeability. Examples of the carbonate used include sodium carbonate and potassium carbonate. Examples of the bicarbonate include sodium bicarbonate and potassium bicarbonate. 1-10 mass% is preferable, as for the density | concentration of carbonate aqueous solution or bicarbonate aqueous solution, 2-8 mass% is more preferable, and 3-7 mass% is further more preferable.
The treatment with the carbonate aqueous solution or the bicarbonate aqueous solution may be performed by dropping these aqueous solutions at room temperature onto the sample.

Step (2) is a step of treating with a mordant test solution containing iodine or picric acid.
Examples of the mordant test solution containing iodine include an iodine-potassium iodide aqueous solution and an iodine-sodium hydroxide aqueous solution. The iodine concentration in the mordanting test solution is preferably 0.1 to 0.3% by mass, and more preferably 0.15 to 0.25% by mass.
Examples of the mordant test solution containing picric acid include picric acid-ethanol solution. The picric acid concentration in the mordant test solution is preferably 1-2% by mass.

  In step (2), for example, a mordant test solution may be added to the sample and reacted at room temperature for 30 seconds to 1 minute. After the reaction, the sample is washed with water.

Step (3) is a step in which the sample is treated with a decolorization reagent containing a polar organic solvent.
Examples of the polar organic solvent include alcohols, ketones, and mixed solvents thereof. As alcohol, C1-C4 alcohol, for example, ethanol, propanol, isopropanol, etc. are mentioned. Examples of ketones include acetone and methyl ethyl ketone. Examples of the mixed solvent include ethanol-acetone mixed solutions. Further, the ethanol or acetone solution may be an aqueous solution of 80% or more.

  In the step (3), a polar organic solvent may be added to the sample and reacted at room temperature for 30 seconds to 1 minute. After the reaction, wash with water.

  Step (4) is a step of treating the sample with the post-staining reagent of the present invention. In the step (4), for example, a post-staining reagent is added to the sample, and the reaction is performed at room temperature for 30 seconds to 1 minute. After the reaction, wash with water.

According to the Gram staining method of the present invention, Helicobacter spp.
Bacteria such as Campylobacter spp., Legionella spp., Haemophilus spp., Anaerobes can be clearly stained.

Example 1 and Comparative Example 1
(1) Preparation of sample A Helicobacter pylori (pure culture) strain, which is said to be difficult to dye by Gram staining, was smeared on a slide glass and allowed to dry naturally, and then a smear sample fixed with flame was used as a sample.

(2) Preparation of test solution (Pre-staining test solution)
Faber G “Nissui” staining solution A (0.2% Victoria Blue solution) (Nissui Pharmaceutical) was used.

(Mordant, decolorizing reagent)
Faber G “Nissui” decolorization solution (2% picric acid ethanol solution) (Nissui Pharmaceutical product) was used.

(Post-staining reagent)
The test solution shown in Table 1 was used.

  As a comparative example, Faber G “Nissui” decolorizing solution B (0.04% fuchsin solution) (Nissui Pharmaceutical) was used.

(3) Staining treatment 1) The pre-staining test solution was fully loaded on the sample, dyed for 1 minute, and then gently washed with running water.
2) The mordanting and decoloring reagent solution was fully loaded on the sample, and after the reaction for 1 minute, it was gently washed with running water.
3) The post-staining test solution was fully loaded on the sample, dyed for 1 minute, and then gently washed with running water.
4) After natural drying, the sample was microscopically examined at a magnification of 1000 times.

(4) Results The staining results are shown in FIGS.

As is apparent from FIGS. 1 and 2, in the stained image of Helicobacter pylori, which is said to be a difficult-to-stain bacterium by Gram staining, Example 1 is stained more vividly than Comparative Example 1;
The characteristic helical form shown by Helicobacter pylori was clearly confirmed.

Example 2 (pH of post-staining test solution)
In the post-staining test solution of Example 1, Helicobacter pylori was gram-stained by the same operation except that the pH adjuster (HEPES) was changed. The results are shown in Table 2.

  From Table 2, the dyeability improved by adjusting the pH of the post-dyeing test solution to a range of 4.5 to 7.5.

Further, even when the pH is 4.5 to 7.5, when a buffer is used, the pH is stable and stable dyeability is obtained.
Furthermore, the post-staining test solution was stable for 4 months due to the addition of a chelating agent, and precipitation or the like did not occur.

Example 3 (Fuchsin concentration in post-staining reagent)
In the post-staining test solution of Example 1, the fuchsin concentration was changed, and the staining property of Helicobacter pylori was examined. As a result, as shown in Table 3, the fuchsin concentration was in the range of 0.005 to 0.2% by mass, and the dyeability was good.

Example 4 (alcohol concentration in post-staining reagent)
In the post-staining test solution of Example 1, the ethanol concentration was changed and the staining property of Helicobacter pylori was examined. As a result, as shown in Table 4, good dyeability was obtained at an ethanol concentration of 10 to 40% by mass.

Example 5 (type and content of chelating agent in post-staining reagent)
In the post-staining test solution of Example 1, the stainability of Helicobacter pylori was examined by changing the type and concentration of the chelating agent. As a result, as shown in Table 5, good dyeability was obtained regardless of the type of chelating agent.

Claims (6)

  A post-staining test solution for Gram dyeing containing (A) basic fuchsin, (B) a lower alcohol, and (C) a base having a pH of 4.5 to 7.5.   The post-dyeing reagent solution for Gram dyeing according to claim 1, further comprising (D) a chelating agent.   The post-dyeing reagent solution for Gram dyeing according to claim 1 or 2, wherein the component (A) is basic fuchsin.   The post-dyeing reagent solution for Gram dyeing according to claim 2 or 3, wherein (D) the chelating agent is a chelating agent selected from aminopolycarboxylic acids, aromatic or aliphatic carboxylic acids, phosphonic acids and hydroxycarboxylic acids.   (1) a step of treating a sample with a pre-staining reagent containing crystal violet or Victoria Blue, (2) a step of treating with a mordant reagent containing iodine or picric acid, and (3) a decoloring reagent containing a polar organic solvent. A Gram dyeing method comprising: a step of treating; and (4) a step of treating with the post-gram dyeing reagent for Gram dyeing according to any one of claims 1 to 4.   The Gram dyeing method according to claim 5, further comprising a step of treating with an aqueous carbonate solution or an aqueous bicarbonate solution between the step (1) and the step (2).