Documentation

Introduction

Basic Fuchsin is a fluorescent dye containing a mixture of rosaniline, pararosaniline, magenta II, and new fuchsine. The basic fuchsin is a cationic triphenylmethane dye used for the detection of acid-fast bacilli and is commonly used in the Ziehl Neelsen staining technique. It stains mucopolysaccharides and glycoproteins. It can also be used to track proteins in acidic pH systems. The basic fuchsin stain acts as a nuclear stain, elastic tissue stain, and mucin stain.

The basic fuchsin dye is dominantly used in staining procedures. It is used for Gomori’s aldehyde-fuchsin method, periodic acid Schiff method, and in Endo medium to distinguish E.coli from K.aerogenes bacteria. The basic fuchsin is also employed as a counter-stain in Gram’s staining.

Properties

A mixture of pararosaniline, rosaniline, new fuchsine, and magenta II makes basic fuchsin dye. Its chemical and physical properties are mentioned below:

1. Chemical Name: 4-[bis(4-aminophenyl)methylidene]cyclohexa-2,5-dien-1-iminium chloride
2. Molecular Formula: C₁₉H₁₈ClN₃
3. Molecular Weight: 824 g/mol
4. Melting Point: 268-270^oC
5. Color: Dark Green
6. Odor: Odorless
7. Solubility: Soluble in water
8. Physical appearance: Green crystalline powder
9. Density: 1.22g/cm³

Staining methods

The Basic fuchsin stain is used in a variety of staining methods including the Ziehl Neelsen technique, Taylor’s protocol, aldehyde fuchsin method, Mowry’s aldehyde fuchsin modification method, PAS method, and the Feulgen staining procedure. All these staining techniques have basic fuchsin in one of the solutions. The protocols followed for these staining techniques are discussed below:

Ziehl Neelsen Staining

This procedure is used to stain acid-fast bacilli. The mycolic acid in the cell membranes of the acid-fast bacilli retains the carbon fuchsin dye in this staining technique. The carbol fuchsin dye makes the bacteria appear red under the microscope. The staining protocol is described below:

1. Prepare the glass slide with the specimen.
2. Fix the sample with the help of heating or alcohol.
3. Place carbol fuchsin over the smear. Heat the slide for 5 minutes until fumes appear (Do not overheat).
4. Pour 20% sulfuric acid and wait for 1 minute. Repeat the step until pinkish-red color appears.
5. Pour methylene blue and wait for 2 minutes and then wash the slide with water.
6. Dry the slide and observe it under the microscope.

The red-stained acid-fast bacilli with a blue background were analyzed under the microscope.

Taylor’s method

This staining technique is used for Gram-negative and Gram-positive bacteria. The staining procedure for Taylor’s method is described as follows:

1. Deparaffinize and hydrate the slides.
2. Add 1ml crystal violet (1%), and 5 drops of sodium bicarbonate (5%) in a vial and mix. Pour the mixture onto the slides and stain the slides for 1 minute, then rinse with distilled water.
3. Place Gram’s iodine on the slides for 1 minute and rinse with water, blot dry the slides after iodine staining.
4. Decolorize the slides with acetone.
5. Stain the slides in basic fuchsin for 1 minute and wash them. Do not dry the slides entirely during blot drying.
6. Differentiate the specimen by dipping the slides in acetone, then transfer the slides immediately to the picric acid solution. Repeat the process until sections appear yellowish-pink.
7. Rinse the slides with acetone quickly.
8. Mount the slides on the microscope for observation.

The Gram-positive bacteria appear blue (filaments), while Gram-negative bacteria look red (nucleus), and additional tissue elements appear yellow under the microscope.

Aldehyde-fuchsin method

This staining technique is used to stain elastic fibers and pancreatic islet beta cells. The method follows the protocol mentioned below:

1. Deparaffinize and hydrate the sample slides.
2. Place the slides in 70% alcohol solution for 30 seconds.
3. Stain the slides in aldehyde fuchsin solution for 30-60 minutes.
4. Rinse the slides with four changes of distilled water.
5. Place the slides in a fast-green solution for 1 minute.
6. Rinse the slides with three changes of distilled water.
7. Dehydrate in graded alcohols.
8. Clear the slides in three or four changes of xylene solution.
9. Mount the slides on the microscope with synthetic resin.

The pancreatic beta cells appear purple to violet with a green background under the microscope. The elastic tissues also look purple to violet after staining with the aldehyde-fuchsin solution.

PAS method

The PAS stain uses periodic acid which oxidizes the carbon-carbon bond forming aldehydes. These aldehydes then react to fuchsin-sulfurous acid making a magenta color. The basement membranes, glycogen, undifferentiated squamous cell carcinoma, and mucosubstances secreted from the epithelia of different organs are distinguished and demonstrated using the PAS method. Kidney and liver biopsies also use the PAS staining technique. The procedure is discussed below:

1. Deparaffinize and hydrate the slides with distilled water.
2. Keep the slides in 0.5% picric acid for 5 minutes.
3. Rinse the slides with distilled water.
4. Place the slides in Schiff’s reagent for 40-50 seconds and microwave them until a deep magenta color appears.
5. Wash the slides in running tap water for 5 minutes.
6. Counterstain with hematoxylin for 3 minutes.
7. Wash with tap water.
8. Dehydrate the slides with alcohol.
9. Mount the slides on the microscope for analysis.

The fungus and glycogen appear magenta with blue stained nuclei.

Feulgen method

The Feulgen method is used to specify chromosomes and DNA in nuclei. The method employs Schiff’s reagent for staining. The protocol followed in this technique is described below:

1. Deparaffinize the slides using xylol. Using 100%, 95%, 70% and 50% alcohols rehydrate the slides.
2. Using distilled water rinse the slides.
3. Pass the slides sequentially through the following steps:
   1. Extract one slide in 1N HCl at 60° C (12 min.)
   2.  
   3. Extract the second slide in 1N HCl at 60° C (1 hour)
   4. Extract the third slide in water at 60° C (1 hour)
   5. Rinse gently with fresh distilled water (1 min.)
   6. Stain in Schiff’s reagent (30 min.)
   7. Bleach with sulfurous acid (10 min.)
   8. Rinse gently with fresh distilled water (1 min.)
   9. 70% alcohol (5 min.)
   10. 100% alcohol (5 min.)
   11. Xylol (5 min.)
4. Mount the slides on the microscope for histological analysis.

Applications

The basic fuchsin staining technique can be used for histological and morphological analysis of a variety of human tissues, surgical specimens, and plant tissues. Also, the staining method is the most promising staining technique used for the examination of acid-fast bacteria, gram-positive and gram-negative bacteria, hepatitis B surface antigen (HBsAg), and sections of the pancreas, kidney, liver, and small intestine. Following are some of the research applications of the basic fuchsin staining method.

The use of basic Fuchsin in Plant Anatomy

Camp and Liming utilized the basic fuchsin dye to trace the vascular bundles of herbaceous plants and the water-conducting system of woody plants. In the experiment, a stock solution of 2.5% basic fuchsin in 95% alcohol was used. The tissue specimens were observed under the microscope for histological and morphological examination. The basic fuchsin dye enabled the researchers to analyze plant anatomy by increasing the contrast in the sample slides.

Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon

In the study, the basic fuchsin dye was used to visualize lignin in the basal internodes of three Brachypodium distachyon ecotypes displaying different flowering times and analyze them using the fluorescent imaging technology. With the help of the basic fuchsin staining, the subcellular patterns of lignin in vascular and interfascicular fiber cell walls were visualized. The basic fuchsin dye did not quantify the lignin content in the subcellular compartments but provided the researchers with a new comparative tool for lignin imaging. This research also indicated that the Basic Fuchsin fluorescence is directly proportional to the amount of lignin between samples, stains hydroxycinnamates, and maybe inconsiderate to lignin composition.

Banding of human chromosomes with basic fuchsin

Scheres and Merkx (1976) used the basic fuchsin stain to perform human metaphase chromosome banding. The cationic triphenylmethane dye specified the G-banding pattern’s main characteristics: the secondary constriction regions of chromosomes No. 1 and 16, the heterochromatic area of chromosome No. 9, and a number of chromosomal regions with bright Q fluorescence, the polymorphic regions of chromosomes No. 3, 4, and Y clearly. The research indicated that the basic fuchsin staining technique is suitable for telomeres, family, and linkages study.

Basic Fuchsin as a Nuclear Stain

The basic fuchsin dye is an excellent stain to study the nuclei of fungi. In this study, aqueous basic fuchsin was tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi. Formaldehyde acted as a mordant to stain the samples permanently. The results suggested that the basic fuchsin is the most promising stain for the morphological and chemical analysis of fungi nuclei.

Strengths and limitations

The basic fuchsin has been widely used for staining purposes in biomedical research. Following are the advantages and weaknesses of the basic fuchsin staining.

1. The basic fuchsin staining method is a safe, reliable, and efficient staining technique used in biomedical research.
2. The basic fuchsin dye is used in many staining procedures: Ziehl Neelsen technique, Taylor’s protocol, aldehyde fuchsin method, Mowry’s aldehyde fuchsin modification method, PAS method, and the Feulgen method.
3. The basic fuchsin staining technique can be used to visualize elastic fibers and cardiac and skeletal muscle tissue sections.
4. Basic Fuchsin can be used to stain tubercle Bacillus, and bacterial flagella, and to compare Escherichia coli and Aerobacter aerogenes.
5. The dye has been useful in understanding micro-fissures, as it allows a clear demonstration of surface-level structures with a constant background.

Precautions

Following precautionary measures should be taken while handling the basic fuchsin dye.

1. Avoid breathing as basic fuchsin is toxic.
2. Wear proper gloves and masks for handling.
3. Avoid eye contact because it can cause irritation.
4. If inhaled keep the victim in fresh air for some time.
5. If touched, wash the exposed part immediately.
6. Keep the dye away from heat as it is flammable.
7. Dispose of the waste properly after the experiment.

References

1. Delamater, E. D., & Ulrich, D. (2009). Basic Fuchsin as A Nuclear Stain. Stain Technology, 161-176.
2. Kapp, N., Barnes, W. J., Richard, T. L., & Anderson, C. T. (2015). Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon. Journal of Experimental Botany, 4295–4304.
3. Scheres JM, M. G. (1976). Banding of human chromosomes with basic fuchsin. Hum Genet, 32(2), 155-69.
4. W.H.Camp, & F.G.Liming. (2009). The use of basic Fuchsin in Plant Anatomy. Stain Technology, 91-9.