GRAM STINKING METHOD

Description

[0001] Gram staining is a fundamental laboratory technique used to distinguish and classify microorganisms, particularly bacteria, into Gram-positive and Gram-negative categories. Developed in 1884 by the Danish bacteriologist Hans Christian Gram, this staining method relies on the ability of bacterial cell walls to retain or lose a violet dye during a series of staining steps. Gram-positive bacteria retain the dye due to their thick peptidoglycan layer and appear violet to dark blue under the microscope, while Gram-negative bacteria lose the dye due to the specific architecture of their cell wall, which consists of an outer membrane and only a thin peptidoglycan layer. Upon counterstaining, they appear pink to rose.Gram staining is an indispensable tool in microbiology, which helps in the identification and classification of microorganisms, especially bacteria, and in guiding treatment decisions in the clinical setting.

[0002] The Gram staining method encompasses both qualitative and quantitative aspects, with numerous factors influencing the final result, including sample thickness, washing methods, dye concentrations, and decolorization techniques. Despite the variability caused by these factors, the method remains robust and allows for some flexibility in techniques while maintaining precision within defined limits.

[0003] Today, the Gram staining method is widely recognized as a significant contribution to clinical diagnostics and research. In bacteriology, it represents the first and extremely valuable step in diagnosis and classification. The ability to bind the primary dye in Gram staining is a unique property found in only a small subset of natural materials. Most plant and animal cells exhibit Gram-negative staining. Gram-positive staining is primarily found in yeasts, bacteria, and molds. Some other materials, such as cell nuclei, certain protozoa, keratohyalin, and viral proteins, have been identified as weakly to strongly Gram-positive.

[0004] In the standard Gram staining procedure, after staining microorganisms, especially bacteria, with a basic aniline dye (usually crystal violet), a dye-iodine complex is formed within the bacteria by subsequent treatment with Lugol's solution (which contains an iodine-potassium iodide complex). This dye complex, unlike the primary dye, is insoluble in water. However, it is soluble in ethanol and is therefore extracted from Gram-negative bacteria by treatment with ethanol. Due to their thicker peptidoglycan layer, it is not extracted from Gram-positive bacteria under Gram staining conditions using ethanol.

[0005] According to current technology, the dyeing process consists of three steps: 1. Dyeing:

[0006] In the first step, a solution of crystal violet (gentian violet) to which 15 g / l of phenol (carbolic acid) has been added, the so-called "carbolic gentian violet," is used. This stains all microorganisms / bacteria, both gram-positive and gram-negative. In the subsequent treatment with Lugol's solution, larger dye complexes are formed, and all microorganisms / bacteria appear dark blue. 2. Decolorization ("differentiation"):

[0007] The second step involves treatment with 96% ethanol. Gram-positive and gram-negative microorganisms / bacteria behave differently in this process: gram-negative microorganisms / bacteria are quickly decolorized, while the blue dye complexes from gram-positive microorganisms / bacteria can only be washed out after significantly longer treatment with ethanol. 3. Counterstaining:

[0008] To visualize the gram-negative microorganisms / bacteria, they can be counterstained with diluted fuchsin solution or safranin solution, whereupon they appear red or reddish-orange respectively.

[0009] According to current technology, treatments with Lugol's solution and with alcohol are the crucial steps in Gram staining.

[0010] According to current technology, the Gram staining method uses several reagents that can be potentially hazardous substances. These reagents include crystal violet, Lugol's solution (iodine-potassium iodide), ethanol or acetone, and safranin. Potential hazards include skin irritation, eye and respiratory irritation, and the flammability of solvents such as ethanol or acetone contained in the staining reagents. It is crucial to observe appropriate safety precautions when handling these reagents, such as wearing personal protective equipment like gloves and safety goggles, and working in well-ventilated areas.

[0011] WO 2001 / 088177 teaches a staining solution for bacteria that does not contain phenol. The staining solution contains 4-methoxyphenol, 2-phenylethanol, benzyl alcohol, 2-butoxyethyl acetate, or cyclohexanol instead of phenol.

[0012] BURKE, VICTOR, "NOTES ON THE GRAM STAIN WITH DESCRIPTION OF A NEW METHOD", JOURNAL OF BACTERIOLOGY, Vol. 7, No. 2, March 1, 1922, discloses a Gram staining method in which crystal violet is used as the primary stain, Lugol's solution as the mordant, a decolorizing solution containing no alcohols, and safranin as the counterstain. Furthermore, all these reagents are used in aqueous solution, and the sample is washed with distilled water after each addition of reagents.

[0013] N. Kopeloff & P. ​​Beerman, "Modified gram stains", J. Infectious Diseases, November 30, 1922, describe a similar Gram staining procedure using fuchsin as a counterstain. In addition, the sample is not rinsed with water after each reagent addition in this procedure.

[0014] US 5,827,680 A discloses a variant of the original Gram staining method that includes counterstaining (a combination of safranin and fuchsin) in the decolorizing solution (a mixture of acetone and isopropanol). The dyes are dissolved in ethanol.

[0015] JP 2003 169694 A discloses a variant of the classical Gram staining method using decolorizing solutions containing alkanols.

[0016] JP H10 236914 A discloses a dyeing composition containing fuchsin (or safranin) dissolved in diethylene glycol monoethyl ether.

[0017] The objective underlying this invention was to provide a method for Gram staining which does not require alkanols, in particular methanol, ethanol and isopropanol, as solvents and thereby reduces the risk of ignition.

[0018] The development of alkanol-free reagents for the Gram staining method offers significant advantages in terms of safety for people and environmentally friendly handling when using this staining technique in microbiology.

[0019] The invention relates to a method for staining microorganisms, in particular bacteria, comprising the steps a) Staining a bacterial sample with a solution of crystal violet, b) Adding Lugol's solution to the stained bacterial sample, c) Rinsing with distilled water, d) Adding a solution of safranin O and fuchsin in diethylene glycol monoethyl ether, e) Rinsing with distilled water the process is essentially alkanol-free.

[0020] The reagents used for Gram staining are essentially alkanol-free, and in particular, essentially ethanol-free. The term "essentially alkanol-free" means that the alkanol content of the crystal violet and safranin solutions, as well as Lugol's solution, is less than 1 vol%, preferably less than 0.1 vol%. Likewise, the alkanol content of any rinsing solutions used is less than 1 vol%, preferably less than 0.1 vol%. During the execution of the process, the alkanol concentration remains below 1 vol%, preferably less than 0.1 vol%, at all times. In particular, "alkanol" refers to methanol, ethanol, and isopropanol.

[0021] Bulk densities for coloring pigments, such as crystal violet, safranin-O, or fuchsin, are generally in the range of 0.7 to 0.8 g / cm³, preferably 0.75 g / cm³. For the purposes of this invention, a conversion from volume percent to weight percent is performed using a bulk density of 0.75 g / cm³. The bulk density of ammonium oxalate is 0.48 g / cm³. The bulk density of potassium iodide is 1.5 g / cm³.

[0022] Values ​​given in vol.%, in particular the volumes of reagents 1, 2 and 3 (see Tables 1, 2 and 3), refer to the total volume of the composition in question as 100 vol.%. Vol.% values ​​for the process refer to the total liquid volume in the respective process step.

[0023] Besides the reduced risk of inflammation, a further advantage of the invention lies in the fact that a separate decolorization step, which in the prior art is usually carried out with pure ethanol, is eliminated. In the process according to the invention, staining is first carried out with a mixture of crystal violet and Lugol's solution in steps a) and b), and then a combined destaining and counterstaining with a safranin / fuchsin solution is carried out in step d).

[0024] The crystal violet solution (also called Reagent 1), used to stain the bacterial sample in step a, contains, in addition to crystal violet (CI 42555), a solvent, preferably water and dimethyl sulfoxide (DMSO). This solution also contains a pH adjuster, preferably ammonium oxalate and sodium bicarbonate. The preferred composition of Reagent 1 is shown in Table 1. Table 1: Preferred composition of reagent 1 for the Gram staining method Reagent 1: Crystal violet solution minimum purity Example proportion (vol.-%) Possible proportion (vol.%) Part A DMSO ≥ 87 % 20 20 - 70 Part B Diammonium oxalate monohydrate ≥ 99 % 10 1 - 10 Part C Sodium bicarbonate ≥ 99 % 12,5 1 - 12,5 Part D Crystal Violet (CI: 42555) ≥ 90 % 10 1 - 20 Component E distilled water distilled water 47,5 30 - 77

[0025] In Table 1, the column "Possible proportion" gives the general composition of reagent 1. This applies accordingly to the entries in the following tables.

[0026] The Lugol's solution, or reagent 2, with which the stained bacterial sample is treated in step b), preferably has the composition shown in Table 2, which is in accordance with the prior art. Table 2: Composition of reagent 2 for the Gram staining method. Reagent 2: Lugol's solution minimum purity Example proportion (vol.-%) Possible proportion (vol.%) Part A Potassium iodide ≥ 99 % 10 1 - 30 Part B Poly(vinylpyrrolidone)-iodine complex - 25 1 - 30 Part C distilled water distilled water 65 40 - 98

[0027] In step d, decolorization and countercoloration occur simultaneously. Decolorization is achieved by component A from reagent 3, i.e., diethylene glycol monoethyl ether. Countercoloration is achieved with components B and C (safranin-O and fuchsin). Reagent 3 is a solution of safranin-O and fuchsin in diethylene glycol monoethyl ether. The preferred composition of reagent 3 is shown in Table 3. Table 3: Composition of reagent 3 for the Gram staining method. Reagent 3: Decolorizing / countercoloring solution minimum purity Example proportion (vol.-%) Possible proportion (vol.%) Part A Diethylene glycol monoethyl ether ≥ 78 % 97,54 77,5 - 99,4 Part B Safranin-O (CI: 50240) ≥ 80 % 2,1 0,5 - 10 Part C Basic Fuchsia ≥ 80 % 0,36 0,1 - 12,5

[0028] Between staining steps b) and d) (as shown in Table 4), rinsing with distilled water is performed to remove excess staining reagent. Particularly when staining with crystal violet, it is necessary to perform a rinse after the specified incubation time to prevent damage to the bacteria due to excessive incubation.

[0029] In the process according to the invention, staining steps a), b), and d), as well as rinsing steps c) and e), are carried out (as shown in Table 4). In the first step, the sample is stained with crystal violet, in the second step it is stabilized with Lugol's solution, and then briefly rinsed with distilled water. In step d), the stained sample is simultaneously decolorized and counterstained with a solution containing safranin O and fuchsin. After counterstaining, the samples must be rinsed again with distilled water.

[0030] The staining method according to the invention proceeds in a precise sequence (1-5) with predetermined incubation times to ensure its functionality. Table 4 shows the primary steps of the Gram staining method (these are the staining steps a), b), and d), as well as the rinsing steps c) and e). Table 4: Steps of the Gram staining method and reagents Steps Reagent designation 1. a): Primary staining Crystal violet solution 2. b): Stabilization / Pickling Lugol's solution 3. c): Flushing distilled water 4. d): Decolorization / countercoloration Saffron / Fuchsin Solution 5. e): Flushing distilled water

[0031] Incubation times can vary considerably. Table 5 shows incubation times considered as a basic timeframe (functional time) and advantageous duration (recommended time). Additionally, there are various incubation ranges that encompass the spectrum of possible times within which the reactions with the reagents can be successfully carried out. Table 5: Incubation times of the Gram staining method Protocol with incubation times Steps reagent recommended time (mm:ss) functional time (mm:ss) A Crystal violet incubation 03:00 00:05 - 10:00 B Incubation with Lugol's solution 01:00 00:05 - 10:00 C Rinse with distilled water 00:05 00:05 - 05:00 D Saffron-fuchsin incubation 00:25 00:20 - 10:00 E Rinse with distilled water 00:30 00:05 - 10:00

[0032] The following describes the composition of the reagents to be used in the process according to the invention. Reagent 1: Crystal violet solution

[0033] Table 6: Component names and list of abbreviations for the preparation of a crystal violet solution Component # Component name abbreviation Component 1 Crystal Violet (CI: 42555) CV Component 2 Sodium bicarbonate SBC Component 3 Diammonium oxalate monohydrate DAOx Component 4 Distilled water DW Component 5 Dimethyl sulfoxide DMSO Reagent 2: Lugol's solution

[0034] Table 7: Component names and list of abbreviations for Lugol's solution (reagent 2) Component # Component name abbreviation Component 1 Potassium iodide AI Component 2 Distilled water DW Component 3 Polyvinylpyrrolidone-iodine PVP-I Reagent 3: Decolorizing / countercoloring solution

[0035] Table 8: Component names and list of abbreviations for the preparation of a decolorizing / countercoloring solution Component # Component name abbreviation Component 1 Saffron-O SF Component 2 Basic Fuchsia BF Component 3 Diethylene glycol monoethyl ether DEME

Claims

1. Method for staining microorganisms, comprising the steps of a) staining a microorganism sample with a solution of crystal violet, b) adding Lugol's solution to the stained bacterial sample, c) rinsing with distilled water, d) adding a solution of safranin-O and fuchsine in diethylene glycol monoethyl ether, e) rinsing with distilled water, wherein the alkanol concentration is less than 1% by volume, preferably less than 0.1% by volume, at all times while the method is being carried out.

2. Method according to claim 1, wherein the solution of crystal violet contains water, dimethyl sulphoxide or a mixture thereof as a solvent.

3. Method according to claim 1 and / or claim 2, wherein the solution of crystal violet contains ammonium oxalate, sodium bicarbonate or both as a pH adjusting agent.

4. Method according to claim 2, wherein the amount of dimethyl sulphoxide is 20-70% by volume, and the amount of water is 30-77% by volume.

5. Method according to claim 3, wherein the amount of ammonium oxalate is 1-10% by volume, the amount of sodium bicarbonate is 1-12.5% by volume, and the amount of crystal violet is 1-20% by volume.

6. Method according to one or more of claims 1 to 5, wherein the Lugol's solution contains water, potassium iodide and a polyvinylpyrrolidone-iodine complex.

7. Method according to claim 6, wherein the amount of water is 40-98% by volume, the amount of potassium iodide is 1-30% by volume, and the amount of polyvinylpyrrolidone-iodine complex is 1-30% by volume.

8. Method according to one or more of claims 1 to 7, wherein the solution of safranin-O and fuchsine in diethylene glycol monoethyl ether contains 77.5 to 99.4% by volume diethylene glycol monoethyl ether.

9. Method according to one or more of claims 1 to 7, wherein the solution of safranin-O and fuchsine in diethylene glycol monoethyl ether contains 0.5 to 10% by volume safranin-O or 0.1 to 12.5 % by volume fuchsine.

10. Method according to one or more of claims 1 to 9, wherein the duration of step a), staining a bacterial sample with a solution of crystal violet, is between 5 s and 10 min.

11. Method according to one or more of claims 1 to 10, wherein the duration of step b), adding Lugol's solution to the stained bacterial sample, is between 5 s and 10 min.

12. Method according to one or more of claims 1 to 11, wherein the duration of step c), rinsing with distilled water, is between 5 s and 5 min.

13. Method according to one or more of claims 1 to 12, wherein the duration of step d), adding a solution of safranin-O and fuchsine in diethylene glycol monoethyl ether, is between 20 s and 10 min.

14. Method according to one or more of claims 1 to 13, wherein the duration of step e), rinsing with distilled water, is between 5 s and 5 min.

15. Method according to claim 14, wherein step e) is repeated once.