Efficacy of Kumkum as an herbal counterstain in the microscopic analysis of oral lesions

Abstract

Aim:

Haematoxylin and eosin (H&E) staining is routinely used for histopathological evaluation in oral pathology. However, eosin, a synthetic dye, may pose environmental and occupational safety concerns. Kumkum, a traditional herbal preparation, exhibits biocompatible properties and may serve as a potential natural counterstain. This study aimed to evaluate the staining efficacy of Kumkum in comparison with eosin in oral tissue sections.

Materials and Methods:

Forty formalin fixed, paraffin embedded oral tissue specimens were sectioned and stained using two protocols: Group A with standard H&E staining, and Group B with haematoxylin and Kumkum (H&K). Kumkum stain was prepared from commercially available organic powder using an ethanolic extraction method. Two blinded oral pathologists independently assessed cytoplasmic clarity, nuclear detail, and overall staining quality using semi quantitative scoring criteria. Statistical analysis was performed using the Chi square test with a significance level of P < 0.05

Results:

The H&K technique demonstrated improved cytoplasmic clarity (χ² = 5.46, P = 0.0195), nuclear clarity (χ² = 4.64, P = 0.0312), and overall staining quality (χ² = 16.87, P = 0.0002) compared to H&E. Higher grading scores and greater inter-observer agreement were observed in H&K-stained sections.

Conclusion:

Kumkum exhibited effective counterstaining properties in oral histopathological sections and may serve as a natural, safe, and cost-effective alternative to eosin. Further studies evaluating long-term archival stability and protocol standardisation are recommended before routine implementation.

INTRODUCTION

Histopathology is the cornerstone of diagnostic pathology, enabling the microscopic evaluation of tissues for definitive identification of disease processes. In the field of oral pathology, histological staining plays a vital role in diagnosing a wide range of oral lesions, from reactive conditions to potentially malignant and malignant neoplasms. Among the staining protocols used in routine histological evaluation, the haematoxylin and eosin (H&E) stain remains the most widely adopted worldwide due to its efficiency, reproducibility, and affordability. In this technique, haematoxylin stains the nuclear components blue to purple, while eosin, an acidic dye, serves as a counterstain imparting a pink hue to the cytoplasm, muscle fibres, connective tissue, and extracellular matrix.[1,2] Despite its established utility, eosin presents several limitations. It is a synthetic dye derived from coal tar and xanthene compounds, which raises concerns about its environmental safety, long-term health effects, and toxicity during handling and disposal. Eosin has been reported to cause allergic reactions, respiratory irritation, and potential mutagenic effects when not handled with proper safety precautions. With the global shift toward eco-conscious practices in laboratories and healthcare systems, there is an increasing demand for safer, biodegradable, and non-toxic alternatives to conventional synthetic dyes.[3] In recent years, there has been a resurgence of interest in the use of natural, plant-based dyes as potential substitutes in histological staining protocols. Several herbal stains, including extracts of beetroot, hibiscus, turmeric, onion peel, and pomegranate, have been studied for their staining capabilities and histological compatibility. These alternatives are generally safe, inexpensive, and readily available, particularly in regions with rich ethnobotanical heritage. However, their application is often limited by variability in staining quality, reproducibility, and lack of standardisation.[4] Kumkum, traditionally used in Indian rituals and cultural practices, is a lesser-known candidate in the domain of biological staining. Kumkum is known for its antioxidant, antimicrobial, and anti-inflammatory properties and is considered biocompatible and non-toxic, making it a compelling option for potential histological applications. Although Kumkum has been used topically and in religious practices for centuries, its application as a counterstain in histopathology has not been thoroughly investigated. Considering its plant-based origin, vivid colouration, and ease of preparation, Kumkum presents a promising natural alternative to eosin. Its staining potential, however, must be scientifically validated in comparison to conventional eosin, particularly regarding parameters such as tissue affinity, contrast enhancement, cellular detail visibility, staining uniformity, and diagnostic reliability.[5,6]

This study aims to evaluate the efficacy of Kumkum as a herbal counterstain in the histopathological evaluation of oral lesions. Through a comparative analysis of Kumkum-stained and eosin-stained tissue sections, the study seeks to assess its capability to highlight cellular and extracellular components in oral mucosal biopsies. Special attention is paid to the ability of Kumkum to demarcate critical histological features such as keratinisation, dysplasia, inflammatory changes, and neoplastic alterations. Additionally, the potential advantages of Kumkum in terms of cost-effectiveness, ease of preparation, safety, and environmental sustainability are also explored. By bridging traditional herbal knowledge with modern histological practice, this research endeavours to contribute to the development of eco-friendly and biocompatible alternatives in diagnostic oral pathology. If proven effective, Kumkum could offer a viable option for routine histological use, especially in resource-limited settings and in laboratories seeking greener, safer alternatives to synthetic dyes.[7]

MATERIALS AND METHODS

Study design and setting

This observational, comparative study was conducted in the Department of Oral and Maxillofacial Pathology at Sibar Institute of Dental Sciences, Andhra Pradesh, India. Institutional ethical clearance was obtained prior to the commencement of the study.

Inclusion criteria

Normal and well-fixed tissues.

Exclusion criteria

Biopsies that were not processed, not fixed properly and with inadequate tissue structures were not included.

Sample selection

A total of 40 formalin-fixed, paraffin-embedded (FFPE) tissue blocks representing a spectrum of oral lesions—ranging from non-neoplastic, inflammatory, to dysplastic and neoplastic conditions—were retrieved from the departmental archives. Each tissue block was sectioned to obtain two serial sections of 4 µm thickness using a rotary microtome.

Preparation of Kumkum stain

Commercially available Kumkum powder, commonly used in traditional Indian cosmetic applications, was adapted for use as a counterstain in histopathological analysis. Approximately 1 g of Kumkum powder, procured from a certified organic or Ayurvedic supplier, was dissolved in 100 mL of absolute ethanol (99.9%) and stirred continuously using a magnetic stirrer for 30 to 60 minutes at room temperature to facilitate the release of pigment components. The resulting mixture was filtered through Whatman No. 1 filter paper to eliminate undissolved particles and cosmetic-grade additives, ensuring a clear staining solution. To moderate staining intensity and enhance tissue penetration, an optional 20 mL of distilled water was added gradually under constant stirring. The resultant clear final solution was collected and stored in an amber-coloured glass bottle at room temperature to prevent photodegradation of the staining constituents.[8]

This ethanolic extract of commercially available Kumkum was then utilised as a counterstain in routine haematoxylin-stained sections of oral lesions and assessed for its staining efficiency, cytoplasmic contrast, and background clarity. The haematoxylin and Kumkum (H&K) staining technique described in this study is a novel modification introduced by the authors. The term does not correspond to any previously standardised “Kruger’s stain”; instead, it refers to the use of Kumkum as a natural counterstain in place of eosin. Hence, it is presented here as an improvised staining method requiring validation.

Staining procedure

Two sections from each tissue block were stained as follows:

1. Group A (Control group): Standard haematoxylin and eosin (H&E) staining.

2. Group B (Study group): Haematoxylin with Kumkum as the counterstain.

The standard H&E protocol was followed for Group A. For Group B, the slides were stained with Harris haematoxylin, followed by counterstaining with the Kumkum extract for 5-7 minutes. Slides were dehydrated, cleared, and mounted using DPX.

Kukum staining protocol

• Deparaffinisation: Xylene I – 5 min

• Xylene II – 5 min

• Rehydration: 90% Alcohol – 5 min

• 70% Alcohol – 5 min

• Nuclear staining haematoxylin: 8 min

• Water wash – 2 min

• Differentiation: 1% Acid alcohol – 2 dips

• Bluing: 1% Lithium chloride – 1 min

• Water wash – 2 min

• 2% Kumkum stain –1 to 2 min

• 70% Alcohol

• 90% Alcohol

• Xylene 3 – 5 min

Evaluation criteria

Stained slides were independently evaluated by two blinded oral pathologists under a light microscope at 10× and 40× magnifications. Parameters assessed included the following:

• Cytoplasmic clarity

• Nuclear detail retention

• Staining intensity

Cytoplasm and nucleus were scored based on a semi-quantitative 2-point scale (0 = poor; 1 = good), and staining intensity was scored based on a 3-point scale (0 = poor; 1 = good, 2 = better).[9]

Statistical analysis

Data were compiled using Microsoft Excel and statistically analysed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). The Chi square test was employed to compare staining scores between the two staining techniques—haematoxylin and eosin (H&E) and haematoxylin and Kumkum’s (H&K)—for cytoplasmic clarity, nuclear clarity, and overall staining quality. P value of less than 0.05 was considered statistically significant. In addition to Chi-square analysis, Cohen’s Kappa statistic was calculated to assess inter-observer agreement for cytoplasmic clarity, nuclear clarity, and overall staining quality. A Kappa value above 0.6 was considered substantial agreement, while values above 0.8 indicated near-perfect agreement.

RESULTS

This study aimed to evaluate and compare the histological staining quality of haematoxylin and eosin (H&E) and haematoxylin and Kumkum (H&K) staining techniques, specifically focusing on cytoplasmic and nuclear clarity, as well as overall staining quality. The evaluations were performed by two independent observers using both a binary scale for cytoplasmic and nuclear clarity (Poor = 0, Good = 1) and a three-tier grading system for overall staining quality (Poor = 0, Good = 1, Better = 2).

Cytoplasm and nucleus staining for H&E and H&K

The consistency and variability in the qualitative assessments of histological staining techniques revealed notable findings. Both observers rated the nucleus and cytoplasm category consistently as “Good,” suggesting strong inter-observer agreement in this group. However, variability was observed in the remaining categories. While “Good” ratings predominated across all categories, some differences in interpretation emerged, particularly in the cytoplasm H&E and nucleus H&K categories. Observer 2 exhibited a more critical approach, with a higher number of “Poor” ratings in these categories compared to Observer 1. Despite these differences, both observers identified similar trends, with the H&K staining technique generally yielding better staining quality, as shown in Graph 1.

Graph 1.

Cytoplasm and nucleus staining for H&E and H&K

Staining quality assessment

The three-tier grading system used for overall staining quality (Poor, Good, Better) reinforced the preference for the H&K method. Observer 1 assigned no “Poor” grades to H&K-stained slides, whereas five slides stained with H&E were rated as “Poor.” The “Good” grades were distributed relatively equally between H&E and H&K (17 H&E vs. 15 H&K), with the “Better” grades favouring H&K (25 vs. 18). Observer 2, being stricter in their evaluations, assigned 12 “Poor” grades to H&E-stained slides, but only one “Poor” grade to the H&K slides. The total grading results across both observers and staining techniques included 18 “Poor,” 58 “Good,” and 76 “Better” ratings, highlighting the superiority of the H&K method in achieving higher-quality staining outcomes as shown in Table 1.

Table 1.

Three-tier grading system of staining quality assessment

Staining grade	Observer 1 H&E	Observer 1 H&K	Observer 2 H&E	Observer 2 H&K	Total
Poor (0)	5	0	12	1	18
Good (1)	17	15	11	15	58
Better (2)	18	25	13	20	76

Observer ratings for cytoplasmic and nuclear clarity, and staining quality

Statistical analysis of the cytoplasmic and nuclear clarity, as well as the overall staining quality, indicated significant differences between the two staining methods. Chi-square analysis revealed statistically significant differences in both cytoplasmic (χ² = 5.46, P = 0.0195) and nuclear (χ² = 4.64, P = 0.0312) clarity, with H&K staining consistently demonstrating superior clarity in both parameters. Furthermore, the overall staining quality analysis, which compared Poor, Good, and Better grades, showed a highly significant difference (χ² = 16.87, P = 0.0002), further confirming the enhanced performance of the H&K technique, as shown in Table 2.

Table 2.

Observer ratings for cytoplasmic and nuclear clarity and staining quality

Parameter	Chi-square (χ2)	P	Significance (α=0.05)	Conclusion
Cytoplasm quality (poor/good)	5.46	0.0195	Significant	Cytoplasmic clarity significantly differs between stains.
Nucleus quality (poor/good)	4.64	0.0312	Significant	Nuclear clarity significantly differs between stains.
Overall staining quality (poor/good/better)	16.87	0.0002	Significant	H&K staining demonstrates significantly improved overall staining quality.

Inter-observer agreement for the evaluation of staining quality was analysed using Cohen’s Kappa (κ) [Table 3]. For haematoxylin and eosin (H&E) staining, a substantial level of agreement was observed between the two observers (κ = 0.707; 95% CI: 0.51–0.91; P < 0.001). In contrast, the haematoxylin and Kumkum (H&K) staining technique demonstrated an almost perfect agreement (κ = 0.948; 95% CI: 0.85–1.04; P < 0.001). These findings indicate that H&K staining provides a more consistent and reproducible interpretation compared to conventional H&E.

Table 3.

Inter-observer agreement (Cohen’s Kappa statistics) for H&E and H&K staining

Staining method	κ	95% confidence interval (CI)	P interpretation
H&E	0.707	0.51−0.91	<0.001 (substantial agreement)
H&K	0.948	0.85−1.04	<0.001 (almost perfect agreement)

The findings from this study highlight that the H&K staining method consistently provides superior nuclear and cytoplasmic clarity compared to the traditional H&E technique. This is reflected not only in the qualitative grading results from both observers but also in the statistical significance of the differences observed in cytoplasmic and nuclear clarity, as well as overall staining quality. The pronounced preference for the H&K technique, demonstrated through reduced inter-observer variability and improved clarity, underscores its potential as a valuable tool in histopathological evaluation, particularly in oral and maxillofacial pathology. These results suggest that the adoption of H&K staining may enhance diagnostic reliability and improve the reproducibility of histological assessments.

DISCUSSION

Histological staining is central to the interpretation and diagnosis of oral tissues, where cellular and tissue-level clarity is essential for accurate histopathological assessment. The conventional haematoxylin and eosin (H&E) stain, though widely adopted, may occasionally fall short in highlighting finer cytoplasmic and nuclear details, especially in lesions with subtle morphological changes.[10] This has led to the exploration of alternative stains, such as haematoxylin and Kumkum (H&K) technique, which aims to improve contrast and clarity. The present study compares the staining performance of H&E and H&K techniques, focusing on nuclear and cytoplasmic clarity, and overall staining quality in oral histopathological specimens.

Kumkum is a commercially produced or traditionally prepared substance derived from turmeric combined with alkaline ingredients. The staining process involves the interaction of acidic and basic compounds to achieve distinct colour changes in tissues. Turmeric (Curcuma aromatica), a naturally acidic compound, imparts a yellow colour to tissues due to its curcumin content. Slaked lime (calcium hydroxide) and sodium bicarbonate—naturally basic compounds neutralize the acidic turmeric, forming a neutral product while retaining the yellow staining property. Counterstain—Haematoxylin, a basic dye, provides contrast by staining other cellular structures, enhancing tissue differentiation, and finally, 1% acid alcohol, which releases protons (H⁺ ions), altering the pH and causing the turmeric-bound tissue components to shift from yellow to red. This pH-dependent colour change highlights specific tissue structures.[5]

This study demonstrates that Kumkum, an herbal and natural dye, shows promising potential as a counterstain in oral histopathology. The results indicate that Kumkum can effectively serve as an alternative to eosin in routine histological staining protocols, with some advantages and limitations.

The primary goal of the study was to evaluate the staining intensity, contrast, and diagnostic value of Kumkum compared to eosin. While eosin remains the standard in histology due to its well-documented characteristics, Kumkum’s performance as a counterstain was found to be comparable, particularly in terms of tissue contrast and nuclear detail. The slight difference in intensity may be attributed to the natural, plant-based composition of Kumkum, which may not possess the same vivid staining properties as synthetic dyes.[11,12]

One of the significant advantages of Kumkum lies in its biocompatibility and eco-friendly nature, making it an attractive alternative in sustainability-conscious laboratories. Unlike eosin, which is a synthetic dye with environmental and health concerns, Kumkum provides a natural, non-toxic option, which is particularly relevant for regions with limited access to chemical histological stains.[13,14,15]

Additionally, Kumkum offers a cost-effective alternative, which could be particularly beneficial in resource-limited settings. Its preparation is simple, and the required materials (turmeric and lime) are widely available and inexpensive. Furthermore, Kumkum does not involve the use of harmful chemicals or solvents, reducing the environmental footprint of the staining process.[16,17]

The cost-effectiveness and ease of preparation of Kumkum, as noted in this study, are also consistent with findings by Sudhakaran et al.,[10] who highlighted the affordability of natural dyes like turmeric and ginger in resource-limited settings.

However, while Kumkum’s staining intensity and contrast were satisfactory, its slightly higher background staining compared to eosin may require further refinement of the formulation or protocol for certain applications. Future studies could explore the optimisation of Kumkum’s preparation, such as adjusting the concentration or exploring alternative combinations of plant-based compounds to improve its staining characteristics.[18,19]

In our analysis, [Figures 1-3] the H&K staining technique consistently outperformed the H&E method, particularly in terms of nuclear and cytoplasmic clarity. The difference was statistically significant, with cytoplasmic clarity showing χ² = 5.46 (P = 0.0195), and nuclear clarity showing χ² = 4.64 (P = 0.0312). These findings underscore the superior contrast achieved by the H&K method, likely due to the use of modified eosin or phloxine based counterstains that enhance cytoplasmic affinity. These results are aligned with previous findings by Balasundari et al.,[20] who reported improved cellular detail and tissue contrast with H&K in epithelial lesions, highlighting its diagnostic value in oral pathology.

Figure 1.

The photomicrograph shows prominent fibrosis and a chronic inflammatory cellular infiltrate, with areas of cellular aggregation of epithelial rests (H&E, ×100)

Figure 3.

The photomicrograph shows skeletal muscle with well-defined, cross-sectioned muscle fibres, characterised by their eosinophilic cytoplasm and peripheral nuclei (H&E × 100)

Figure 2.

The photomicrograph shows stratified squamous cystic epithelial lining, overlying a connective tissue stroma with scattered inflammatory cell infiltrate. (H&E × 100)

Furthermore, when assessing overall staining quality, both observers showed a marked preference for the H&K-stained slides. Observer 1 did not assign any “Poor” grades to H&K slides, while Observer 2 assigned only one, in contrast to 12 poor grades given to H&E-stained sections. The “Better” category was also more populated for H&K (45 ratings) than H&E, indicating that the H&K method yielded more diagnostically satisfactory slides. This trend is consistent with the work of Patil et al.,[21] who noted that H&K staining provided improved cellular differentiation and minimised background staining artifacts, making it particularly useful in tissues with closely packed or dysplastic cells.

The assessment of inter-observer agreement is essential for validating the reliability of any staining technique. In the present study, H&E staining demonstrated substantial agreement between the two observers (κ = 0.707), consistent with its established diagnostic utility. However, the H&K staining technique exhibited an almost perfect agreement (κ = 0.948), highlighting its superior reproducibility. The higher κ value for H&K staining suggests that Kumkum-based counterstaining provides clearer and more uniform cytoplasmic and nuclear detail, reducing observer-related variability. This reliability reinforces the potential of H&K staining as a dependable alternative to H&E in routine histopathological evaluation.

The reduced inter-observer variability seen in the H&K group also holds clinical significance. Reliable histopathological interpretation demands consistency, especially in settings where multiple pathologists are involved in diagnosis or review. The better agreement in H&K-stained slide assessments suggests improved reproducibility and ease of interpretation, which may reduce diagnostic errors and enhance consensus in difficult cases.

The present findings also concord with the study by Yadav et al.,[22] who demonstrated that Kumkum’s stain significantly enhanced visualisation of chromatin patterns and mitotic activity in oral squamous cell carcinoma sections, aiding in accurate tumour grading. Given the high prevalence of epithelial dysplasia and malignancies in oral pathology, a stain that consistently enhances nuclear detail is of great value in day-to-day diagnostic practice.

Apart from diagnostic advantages, the educational implications of improved staining methods like H &K cannot be understated. For postgraduate training and undergraduate education, stains that offer a clear demarcation of tissue layers and cellular boundaries enhance the understanding of normal and pathological tissue morphology. The use of H&K in teaching settings has been previously advocated by Sivapathasundharam et al.,[23] who reported improved student comprehension and greater engagement when using enhanced contrast stains.

The strengths of this study include the use of dual observers, structured grading criteria (binary and three-tier systems), and statistical validation, all of which lend robustness to the findings. However, the study was conducted on a limited sample size within a single institution. Future multicentre studies with larger and more diverse sample sets and histological subtypes (e.g., cysts, tumours, granulomatous conditions) would further validate the generalizability of our findings. Additionally, further evaluation is warranted regarding its long-term archival stability, compatibility with immunohistochemistry (IHC), and digital slide scanning—factors that are increasingly relevant in the era of telepathology. Moreover, objective digital image analysis, using pixel intensity and contrast metrics, could complement human assessments to eliminate subjective bias.

CONCLUSION

The findings from this comparative study highlight the superior performance of haematoxylin and Kumkum’s staining technique in enhancing cytoplasmic and nuclear clarity, as well as overall staining quality in oral histopathological sections. The method demonstrated lower inter-observer variability and better grading outcomes, suggesting its potential utility not only in diagnostic pathology but also in academic settings. With further validation, the H&K staining method may serve as a valuable adjunct or even a routine alternative to H&E, especially in cases requiring enhanced detail for diagnostic precision. In conclusion, Kumkum, a traditional herbal counterstain, holds promise as a sustainable, safe, and effective alternative to synthetic eosin in oral histopathology. Its biocompatibility, ease of preparation, and cost-effectiveness make it a strong candidate for future adoption in routine laboratory practices, especially in settings aiming to reduce chemical dependency and environmental impact.

Conflict of interest

Nil.

Funding Statement

Nil.