JP2003169694A - Gram's staining reagent solution, gram's staining reagent kit, and gram's staining method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a Gram's preliminarily-staining reagent solution excellent in storage stability, to provide a Gram's staining reagent kit containing the solution as a constituent reagent, and to provide an improved Gram's staining method. <P>SOLUTION: This Gram's preliminarily-staining reagent solution contains crystal violet and an anionic surfactant. The Gram's staining reagent kit contains the Gram's preliminarily-staining reagent solution as the constituent reagent, wherein the Gram's preliminarily-staining reagent solution contains the crystal violet and the anionic surfactant. The Gram's staining method comprises treating a specimen with the preliminarily-staining reagent solution, then with a mordant reagent solution, further with a decolorizing reagent solution, and finally with a additionally-staining reagent solution, wherein the preliminarily-staining reagent solution contains the crystal violet and the anionic surfactant, the mordant reagent solution contains iodine, the decolorizing reagent solution contains a polar organic solvent, and the additionally-staining reagent solution contains safranine, fuchsine carbolate or basic fuchsine. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pre-staining reagent solution for Gram staining having excellent storage stability, a reagent kit for Gram staining containing the same as a constituent reagent, and an improved method for Gram staining.

[0002]

2. Description of the Related Art The Gram staining method was devised to stain bacteria present in tissues, but the procedure is simple and economical, and if one is skilled in staining procedures and microscopic examination of specimens. Since it is possible to infer the causative agent of infectious disease, it is currently one of the rapid test methods with high clinical value.

As a Gram dyeing method, a hacker (Huck) using Gentian violet carbonate as a pre-dying dye is used.
er) Although the original method is known, this method has problems such as the fact that the pigment gentian violet, a pigment, is crystallized during dyeing, so at present, instead of the carboxylic acid gentian violet, crystal violet is used. A modified hacker method is generally used in which is used as a pre-staining dye.

According to this method, Gram-positive and Gram-negative bacteria in a sample are stained with ammonium oxalate-containing crystal violet solution for 1 minute (pre-staining treatment), and then,
Lugol solution (iodine-potassium iodide solution) is mordanted for several minutes (mordanting treatment) several times, and then decolorized with pure ethanol for 30 seconds to 1 minute until the smeared surface becomes colorless (decolorization treatment).
Then, by further counterstaining (post-staining treatment) with a safranin solution for 1 minute, a gram-positive bacterium stained with magenta from crystal violet and a gram-negative bacterium stained with red from safranin are distinguished. Since the dyeing technique requires skill, there is a problem that the determination result is inaccurate because individual differences occur in the dyeing result.

On the other hand, by adding sodium hydrogencarbonate to the pre-dyeing test solution, the solution is kept basic, and the dyeability of crystal violet (basic dye) is improved.
There is the rtholomew & Mittwer method (Burmy method). This method is to pour 1% crystal violet solution, immediately drop a few drops of 5% sodium hydrogen carbonate solution, stain for 30 seconds (pre-staining treatment), and then add 2% iodine solution ( Mordant with 0.1N sodium hydroxide solution for 30 seconds (mordanting treatment), then decolorize with acetone-ethanol mixture for several seconds until the smeared surface becomes colorless (bleaching treatment), and further with Peifel solution for 10-30
This is a method of distinguishing the gram-positive bacterium from the gram-negative bacterium by performing counterstaining (post-staining treatment) for seconds. Compared with the Hacker modified method, the pre-staining process has one more step, but the overall dyeing time is short, and the processing time is about several seconds because the acetone-ethanol mixed solution is used for the decolorization process, and there is little difference due to the procedure and accurate determination. Has the advantage that However, when the crystal violet solution, which is the pre-staining reagent solution, and the sodium hydrogen carbonate solution are mixed and stored,
It is known that crystals are deposited with the passage of time and the dyeability deteriorates. Therefore, in order to prepare a kit, there was no choice but to employ a method in which these were made into two liquids and mixed appropriately at the time of use. However, in consideration of the automation of Gram's staining, the use of this two-liquefied pre-staining reagent solution was not preferable.

On the other hand, there is a modified version of the Burmy method in which the pre-staining reagent solution is liquefied (trade name: Burmy M stain, manufactured by Muto Kagaku Co., Ltd.), but this method does not stain with Gram-negative bacteria. There are problems such as the presence of bacteria and poor dyeability.

Under these circumstances, the development of a one-part pre-staining reagent solution having good dyeability, excellent storage stability of the pre-staining reagent solution and compatible with automatic equipment, and Gram stain using the same The current situation is that improved methods are desired.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and is a pre-staining reagent solution for gram staining excellent in storage stability, and a gram staining reagent kit containing the same as a constituent reagent. And to provide an improved method of Gram stain.

[0009]

The present invention is an invention of a pre-staining reagent solution for Gram stain which comprises crystal violet and an anionic surfactant.

Further, the present invention is an invention of a reagent kit for Gram staining, which comprises a pre-staining reagent solution for Gram staining containing crystal violet and an anionic surfactant as a constituent reagent.

Further, in the present invention, a sample is treated with a pre-dyeing reagent containing crystal violet and an anionic surfactant, and then with a mordant reagent containing iodine, and then with a polar organic solvent. The invention of the Gram dyeing method is characterized in that treatment is carried out with a decolorizing reagent which comprises: and further a post-dyeing reagent containing safranine, fuchsin carbonate or basic fuchsin.

That is, the inventors of the present invention have conducted extensive studies to achieve the above object, and as a result, by performing Gram dyeing using a pre-dyeing reagent solution for Gram dyeing containing an anionic surfactant, Overcome the disadvantages of Gram's staining method, such as the fact that the pre-dyeing reagent solution is liquefied and cannot be used for automatic equipment, and that when this liquefied pre-dyeing reagent solution is mixed, crystals precipitate and the dyeability deteriorates. However, it is possible to support automatic equipment by using a pre-staining reagent solution that has been liquefied. Also, if this pre-staining reagent solution that has been liquefied is used, storage stability is excellent without crystallization and excellent dyeability. The inventors have found that a dyed image can be obtained and completed the present invention.

Examples of the anionic surfactant used in the present invention include amino acid type anionic surfactants such as N-acyl glutamic acid, N-acyl alanine, N-acyl glycine and salts thereof, for example, alkylbenzene sulfonic acid,
Alkylsulfuric acid, oleic acid, cholic acid, N-dodecanoyl sarcosinic acid, salts thereof, and the like, among them, N-dodecanoyl sarcosinic acid, N-acyl glutamic acid, cholic acid, preferable salts thereof, among others, among others, More preferred is N-dodecanoyl sarcosinate. These may be used alone or in appropriate combination of two or more.

N-acyl glutamic acid, N-acyl alanine and N-, which are mentioned as amino acid-based anionic surfactants
The acyl group in acylglycine may be linear, branched or cyclic, and usually has 6 to 18 carbon atoms, preferably 12 to 18 carbon atoms, and specifically, for example, a hexanoyl group, Heptanoyl group, octanoyl group,
Nonanoyl group, decanoyl group, undecanoyl group, lauroyl group, tridecanoyl group, myristoyl group, pentadecanoyl group, palmitoyl group, heptadecanoyl group,
Examples thereof include stearoyl group, and among them, for example, lauroyl group, myristoyl group, palmitolyl group, stearoyl group and the like are preferable.

The alkyl group in alkylbenzenesulfonic acid and alkylsulfuric acid may be linear, branched or cyclic, and usually has 8 to 18 carbon atoms, preferably 10 to 1 carbon atoms.
4, specifically, for example, n-octyl group, isooctyl group, sec-octyl group, tert-octyl group, neooctyl group, n-nonyl group, isononyl group, se
c-nonyl group, tert-nonyl group, neononyl group, n-decyl group, isodecyl group, sec-decyl group, tert-decyl group,
Neodecyl group, n-undecyl group, isoundecyl group,
sec-undecyl group, tert-undecyl group, neoundecyl group, n-dodecyl group, isododecyl group, sec-dodecyl group, tert-dodecyl group, neododecyl group, n-tridecyl group, isotridecyl group, sec-tridecyl group, tert-tridecyl group Group, neotridecyl group, n-tetradecyl group,
Isotetradecyl group, sec-tetradecyl group, tert-tetradecyl group, neotetradecyl group, n-pentadecyl group, isopentadecyl group, sec-pentadecyl group, tert-
Pentadecyl group, neopentadecyl group, n-hexadecyl group, isohexadecyl group, sec-hexadecyl group, tert
-Hexadecyl group, neohexadecyl group, n-heptadecyl group, isoheptadecyl group, sec-heptadecyl group, te
rt-heptadecyl group, neoheptadecyl group, n-octadecyl group, isooctadecyl group, sec-octadecyl group,
Examples thereof include a tert-octadecyl group and a neooctadecyl group. Among them, those having 10 to 14 carbon atoms are preferable, and among them, n-dodecyl group is particularly preferable.

N-acyl glutamic acid, N-acyl alanine, N-acyl glycine, alkylbenzene sulfonic acid,
Examples of the salts of alkylsulfuric acid, oleic acid, cholic acid and N-dodecanoylsarcosinic acid include alkali metal salts such as lithium, sodium and potassium, triethanolamine salts and the like, with sodium being preferred.

Specific examples of N-acyl glutamic acid and salts thereof which can be mentioned as amino acid anionic surfactants include, for example, N-coconut oil fatty acid acyl-L-glutamic acid triethanolamine [trade name: Amisoft (R) CT- 1
2, Ajinomoto Co., Inc.], N-lauroyl-glutamic acid triethanolamine [trade name: Amisoft (R) LT
-12, manufactured by Ajinomoto Co., Inc.], N-coconut oil fatty acid acyl-L
-Potassium glutamate [Product name: Amisoft (R) C
K-12, manufactured by Ajinomoto Co., Inc.], N-coconut oil fatty acid acyl
-Sodium L-glutamate [trade name: Amisoft (R) CS-11, manufactured by Ajinomoto Co., Inc.], N-lauroyl
-Sodium L-glutamate [trade name: Amisoft (R) LS-11, manufactured by Ajinomoto Co., Inc.], myristoyl
-L-Glutamate sodium [trade name: Amisoft (R) MS-11, manufactured by Ajinomoto Co., Inc.], N-myristolyl-L-glutamate potassium [trade name: Amisoft (R) MK-11, Ajinomoto Co., Inc. ], N-stearoyl-L-glutamate disodium [trade name: Amisoft (R) HS-11, Ajinomoto Co., Inc.], N-stearoyl-L-glutamate disodium [trade name: Amisoft (R) HS -11, manufactured by Ajinomoto Co., Inc.], N-coconut oil fatty acid acyl-L-glutamic acid [trade name: Amisoft (R)]
CA, manufactured by Ajinomoto Co., Inc.], N-lauroyl-L-glutamic acid [trade name: Amisoft (R) LA, manufactured by Ajinomoto Co.], N-myristoyl-L-glutamic acid [trade name: Amisoft (R)] MA, manufactured by Ajinomoto Co., Inc.], N-stearoyl-L-glutamic acid [trade name: Amisoft (R) HA,
Ajinomoto Co., Inc.] and the like, and among them, N-coconut oil fatty acid acyl-L-glutamic acid triethanolamine [trade name: Amisoft (R) CT-12, Ajinomoto Co., Inc.], N-lauroyl -Glutamic acid triethanolamine [trade name: Amisoft (R) LT-12, manufactured by Ajinomoto Co., Inc.], N-coconut oil fatty acid acyl-L-glutamate sodium [Product name: Amisoft (R) CS-11, Ajinomoto ( Co., Ltd.], myristoyl-L-sodium glutamate [trade name: Amisoft (R) MS-11, Ajinomoto Co., Inc.] and the like are preferable.

Specific examples of N-acylalanine, N-acylglycine and salts thereof which can be mentioned as the amino acid anionic surfactant include, for example, N-coconut oil fatty acid acylglycillin potassium [trade name: Amilite (R) GCK]. -11
(F), manufactured by Ajinomoto Co., Inc.], N-coconut oil fatty acid acyl-D
L-alanine triethanolamine [trade name: Amilite (R) ACT-12, manufactured by Ajinomoto Co., Inc.] and the like can be mentioned.

More preferred specific examples of the anionic surfactant according to the present invention include sodium N-dodecanoyl sarcosinate, sodium cholate, sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, and tetradecyl sulfate. Examples thereof include sodium, and more preferable examples include sodium N-dodecanoylsarcosinate.

Examples of the pre-dyeing reagent solution for Gram dyeing according to the present invention include those in which an anionic surfactant and crystal violet as described above are dissolved in a lower alcohol-containing aqueous solution.

The concentration of the crystal violet in the pre-staining reagent solution is usually 0.05 to 2 w / v%, preferably 0.2 to 0.6 w.
/ v%.

The concentration of the anionic surfactant in the pre-staining reagent solution is usually 0.02 to 5 v / v%, preferably 0.05 to 0.25 v / v.
%.

The lower alcohol is not particularly limited as long as it dissolves crystal violet and an anionic surfactant, and usually includes ones having 1 to 4 carbon atoms, specifically, for example, methanol and ethanol. , N-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol, and the like, among which methanol, ethanol, and isopropyl alcohol are preferable, and ethanol is more preferable. These may be used alone or in appropriate combination of two or more kinds.

The lower alcohol concentration in the pre-dyeing reagent solution is usually 1 to 40 v / v%, preferably 15 to 25 v / v%.

In addition, it is preferable to appropriately add a buffer to the pre-dyeing reagent solution so that the pH is usually 5.5 to 9.0, preferably 6.0 to 7.0.

Specific examples of the buffering agent include Bis
(2-hydroxyethyl) iminotris (hydroxymethyl)
Methane (Bis-Tris), 2-morpholinoethanesulfonic acid (MES), N, N-bis (2-hydroxyethyl) -2-
Aminoethanesulfonic acid (BES), tris (hydroxymethyl) aminomethane (Tris), piperazine-1,4-
Bis (2-ethanesulfonic acid) (PIPES), 3-morpholinopropanesulfonic acid (MOPS), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TE
Good buffer such as S), phosphate buffered saline (PB
S) and the like, among which Bis-Tris, MES,
BES, PBS and the like are preferred. These may be used alone or in appropriate combination of two or more kinds.

The Gram stain reagent kit according to the present invention comprises:
A pre-staining reagent solution for Gram staining as described above is contained as a constituent reagent, and more specifically, a mordant reagent solution containing iodine, a decolorizing reagent solution containing a polar organic solvent,
And a post-staining reagent solution containing safranine, fuchsin phenate or basic fuchsin as a constituent reagent.

As the mordant reagent containing iodine, those commonly used in this field, for example, Bartholomew & Mit
Preferred are those used in the modified Twer's method, and in the case of preparing this, those obtained by dissolving iodine in a basic aqueous solution such as an aqueous solution of sodium hydroxide can be mentioned.

The concentration of the iodine in the mordant reagent solution is as follows:
Usually 0.1 to 0.3 w / v%, preferably 0.15 to 0.25 w / v%, and basic compounds such as sodium hydroxide are preferably
It is appropriately added so as to be 0.4 to 0.8 w / v%.

Examples of the decolorizing reagent containing a polar organic solvent include alcohols, ketones and mixed solutions thereof.

Examples of alcohols include methanol, ethanol, propanol, isopropanol, 2-
Butanol and the like can be mentioned, with preference given to methanol and ethanol.

Examples of the ketones include acetone, methyl ethyl ketone, γ-butyrolactone, etc., among which acetone is preferred.

The mixed solution of these is preferably a mixed solution of ethanol and acetone.

The concentration of the alcohol contained in the decolorization reagent is usually 40 to 100 v / v%, preferably 60 to 100 v.
/ v%, and the concentration of the ketone is usually 0 to 60
v / v%, preferably 0-40 v / v%.

The post-staining reagent solution containing safranine, fuchsin phenate or basic fuchsin according to the present invention is
For example, safranine solution, fuchsin folic acid solution, Peifel solution, basic fuchsin solution and the like can be mentioned. As these, commercially available products may be used, or those appropriately prepared by a conventional method may be used at a normal concentration.

The post-staining reagent solution is preferably a safranin solution when the sample is a patient sample, and is preferably a fuchsin carbonate, a Peifel solution or a basic fuchsin solution when it is a pure culture. Further, in the case of a sample in which bacteria having weak staining properties such as anaerobic Gram-negative bacillus are present, the Peifel solution is preferable.

The Gram staining method according to the present invention is, for example, as follows.

First, after smearing and drying the sample, the sample fixed with alcohol is treated with a pre-staining reagent solution containing the anionic surfactant according to the present invention and a crystal violet solution at 4-37 ° C. for 0.5-0.5%. After treating for 1 minute, it is treated with a mordant reagent containing iodine for 0.5 to 1 minute at 4 to 37 ° C, and then a decolorizing reagent containing a polar organic solvent for 4 to 37 minutes.
After treatment at 5 ° C for 10 seconds, safranine solution, fuchsin phenate, or basic fuchsin is added to the dyeing test solution.
It suffices to perform the treatment at 37 ° C for 5 seconds to 10 seconds. In addition, it is preferable to appropriately wash the sample with water or the like between these treatments. The obtained stained image is recognized as a Gram-positive bacterium when it is stained with a crystal violet solution-derived color (purple to black-purple), and when it is stained with a safranine solution or a Paifel solution-derived color (red) Is recognized as a Gram-negative bacterium.

The preferred embodiments such as specific examples and concentrations of the constituents in these test solutions are as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

[0041]

EXAMPLES Example 1. [Preparation of sample] Two cases of two pure cultures, which are Gram-positive bacteria that are said to be difficult to stain with Gram stain [Staphylococcus aureus: No. 37,352,
Streptococcus pyogenes: No. Four
1,329] was used to prepare a smear. After drying, an alcohol was fixed and completely dried, and then a dyeing operation was performed.

[0042] [Preparation of test solution] (1) Pre-staining reagent: 20v / v% ethanol aqueous solution                   Crystal violet 0.4v / v%                   N-dodecanoyl sarcosinate Na 0.1v / v%                   Bis-Tris Add to pH 6.2 (2) Mordant reagent: iodine 2%                 Sodium hydroxide solution 0.4% (3) Decolorization test solution: Acetone 30v / v%                 Ethanol 70v / v% (4) Post-dyeing reagent: Paifel liquid (manufactured by Wako Pure Chemical Industries, Ltd.)

[Staining Treatment] (1) A pre-staining reagent solution was fully loaded on the sample and reacted at room temperature for 30 seconds. Then, it was washed gently with water. (2) A mordant reagent solution was loaded on the sample and allowed to react at room temperature for 30 seconds. Then, it was washed gently with water. (3) The sample was decolorized with the decolorizing reagent at room temperature for several seconds, and washed gently with water. (4) A sample of Paifel's solution was loaded on the sample and reacted at room temperature for 10 seconds. It was gently washed with water, air-dried and then examined with a microscope. The results are shown in Table 1. In addition, Staphylococcus aureus No.
The stained image of 352 is shown in FIG.

Comparative Example 1. (Conventional one-liquid method) [Sample preparation] The one prepared in the same manner as in Example 1 was used. [Reagent and Staining Operation] Using "Burmy M Staining" kit (manufactured by Muto Kagaku Co., Ltd.), staining was performed according to the standard operating method described in the instructions attached to the kit. The results are also shown in Table 1. In addition, Staphylococcus aureus No. The stained image of 352 is shown in FIG.

Comparative Example 2 (conventional two-liquid method) [Sample preparation] The sample prepared in the same manner as in Example 1 was used. [Reagent and dyeing operation] Using "Gram dye B & M Wako" kit (manufactured by Wako Pure Chemical Industries, Ltd.), dyeing was performed according to the standard operation method described in the instructions attached to the kit. The results are also shown in Table 1. Also, Staphylococcus aureus N
o. The stained image of 352 is shown in FIG.

[0046]

[Table 1]

[Discussion] Staphylococcus aureus Nos. 37 and 352, Streptococcus pyogenes N, which are obtained in Example 1 and Comparative Examples 1 and 2 and are said to be particularly difficult to stain by Gram's staining
Comparing the stained images of o.41 and No.329, Table 1 and FIG.
As is clear from the results of 3, Staphylococcus aureus No. 37,
In addition, there was almost no difference in the stained images of S. pyogenes No. 41 and No. 329. However, for Staphylococcus aureus No. 352, only the stained image obtained by Comparative Example 1 (conventional one-liquid method) was slightly reddish, and the stained area was small (see FIG. 2), and the judgment was unsuccessful. It turned out to be clear.

On the other hand, the stained image of Staphylococcus aureus No. 352 obtained by Example 1 (two-component method according to the present invention) shows the Staphylococcus aureus No. 352 obtained by Comparative Example 2 (conventional two-component method). 352
It was clearer and more bluish than the stained image.

From these results, by using the pre-staining reagent solution of the present invention or the reagent kit for Gram stain containing the same as a constituent reagent, it is possible to obtain a clearer stained image more easily than the conventional one. I understand.

[0050]

As is apparent from the above, the present invention provides a pre-staining reagent solution for Gram staining which is excellent in storage stability, a reagent kit for Gram staining containing the pre-staining reagent solution for Gram staining,
And an improved method of Gram staining using them. By using the present invention, a clearer dyeing image than conventional Gram's dyeing can be obtained and the storage stability of the pre-dyeing reagent solution can be improved. It has an excellent effect that it can be dealt with.

[Brief description of drawings]

1 is a Staphylococcus aureus No. obtained by the method of Example 1. FIG. 352 is a Gram-stained image of 352.

FIG. 2 Staphylococcus aureus No. obtained by the method of Comparative Example 1 352 is a Gram-stained image of 352.

3 is a Staphylococcus aureus No. obtained by the method of Comparative Example 2. 352 is a Gram-stained image of 352.

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 2G045 AA28 BA11 BA14 BB25 BB29                       BB51 BB54 CB21 FA16 FB11                       GC12                 2G052 AA36 AD32 CA03 CA04 CA48                       DA07 FA05 FA09 FC05 FC06                       FD02 FD18 GA32 JA03 JA04                       JA06 JA07 JA08 JA11                 4B063 QA01 QQ06 QR51 QR66 QS36                       QX01

Claims (10)

[Claims] 1. A pre-staining reagent solution for Gram stain, which comprises crystal violet and an anionic surfactant. 2. The anionic surfactant is alkylbenzene sulfonate, alkyl sulfate, amino acid-based anionic surfactant, oleate, cholate or N-dodecanoyl sarcosinate. The staining reagent solution described in 1. 3. The dyeing reagent solution according to claim 1, wherein the anionic surfactant is sodium N-dodecanoylsarcosinate, sodium N-acylglutamate or sodium cholate. 4. A Gram staining reagent kit comprising a pre-staining reagent solution for Gram staining containing crystal violet and an anionic surfactant as a constituent reagent. 5. A mordant reagent containing iodine, a decolorizing reagent containing a polar organic solvent, and a post-dyeing reagent containing safranine, fuchsin carbonate or basic fuchsin as constituent reagents. Item 4. The reagent kit according to Item 4. 6. The anionic surfactant is alkylbenzene sulfonate, alkyl sulfate, amino acid-based anionic surfactant, oleate, cholate or N-dodecanoyl sarcosinate. Reagent kit according to. 7. The reagent kit according to claim 4, wherein the anionic surfactant is sodium N-dodecanoylsarcosinate, sodium N-acylglutamate or sodium cholate. 8. A sample is treated with a pre-staining reagent containing an anionic surfactant and a crystal violet solution, then with a mordant reagent containing iodine, and then with a decolorizing reagent containing a polar organic solvent. And a post-staining reagent solution containing safranine, fuchsin carbonate, or basic fuchsin. 9. The anionic surfactant is alkylbenzene sulfonate, alkyl sulfate, amino acid-based anionic surfactant, oleate, cholate or N-dodecanoyl sarcosinate. Gram stain method described in. 10. The Gram staining method according to claim 8, wherein the anionic surfactant is sodium N-dodecanoylsarcosinate, sodium N-acylglutamate or sodium cholate.