Abstract
Background:
Hematoxylin and eosin (H&E) remains the cornerstone of diagnostic pathology, but the potential harmful effects, high cost, and environmental pollution have necessitated the exploration of eco-friendly and economical alternatives.
Aims:
This study evaluated and compared the overall staining efficacy, durability, and economic feasibility of H&E, hematoxylin and Allium cepa (H&AC), and hematoxylin and Bixa Orellana (H&BO).
Settings and Design:
The comparative study was conducted in the Department of Oral Pathology and Microbiology, Faculty of Dental Science, Dharmsinh Desai University, Nadiad, Gujarat, over 18 months.
Materials and Methods:
A total of 225 slides, including epithelium, collagen fibers, adipose tissue, salivary gland, and bone, were stained with H&E, H&AC, and H&BO. The overall efficacy evaluation was performed on the basis of staining intensity, clarity, and uniformity.
Statistical Analysis Used:
Staining parameters were graded and statistically analyzed using Chi-square and post hoc Dunn and Bonferroni tests (P < 0.05).
Results:
H&E demonstrated superior staining quality across all parameters, with 98.67% excellent intensity, 100% clarity, and 97.33% uniformity (P < 0.001). H&BO emerged as a viable alternative with consistent performance and high efficacy, particularly in collagen and salivary gland tissues. H&AC, while the most cost-effective and eco-friendly, showed variability and reduced staining precision.
Conclusions:
While H&E remains the benchmark in diagnostic histopathology, H&BO presents a promising eco-conscious alternative. H&AC, despite its affordability, requires refinement for consistent diagnostic use. This study highlights the potential for balancing diagnostic excellence with environmental responsibility in routine staining protocols.
Keywords: Diagnostic pathology, eco-friendly dyes, histopathology, natural staining
INTRODUCTION
Histopathology, the cornerstone of diagnostic pathology, is pivotal in diagnosing diseases by visualizing microscopic tissue structures. Histology uses stains that fall into one of two categories: synthetic stains, which are produced through chemical processes, and natural stains, which are made from organic sources. The hematoxylin and eosin (H&E) stain has traditionally been regarded as the most reliable method for the histological examination of human tissues. Synthetic dyes exhibit remarkable efficacy; however, they pose significant environmental hazards. As a result, research has been performed on creating innovative natural dyes that are biodegradable and beneficial to the environment for use in histological staining.[1]
Synthetic dyes can cause hypersensitivity reactions and pose safety issues at their disposal. Eosin, a synthetic xanthene dye, is a class 3 IARC carcinogen and poses environmental threats. Despite the ecological risks and the rising costs of synthetic dyes, natural alternatives are underutilized in practice.[1,2]
This research studied the staining potential of dried red onion (Allium cepa) skin and seeds of annatto (Bixa Orellana) extracts as histopathological stains. The outer layer of red onion skin is rich in anthocyanins, responsible for the red, purple, and blue colors. Dry papery onion skins were known to be used as a natural dye in the textile industry and for coloring eggs. Sinduri (Bixa Orellana) is a commercially important plant grown for its natural dye, annatto, derived from seed arils. The main carotenoids responsible for the yellow to orange color are trans bixin and cis bixin.[3,4,5,6,7,8]
This study compared the staining properties of H&E, Allium cepa (red onion), and Bixa Orellana (annatto) by evaluating the overall staining efficacy of these stains. It also compared the economic feasibility and durability of Allium cepa and Bixa Orellana as natural substitutes for eosin and H&E.
MATERIALS AND METHODS
The comparative study was conducted in the Department of Oral Pathology and Microbiology, Faculty of Dental Science, Dharmsinh Desai University, Nadiad, Gujarat, over 18 months. The institutional ethics committee approved the study bearing the number FDS/DDU/EC/04/2023. Two organic components were collected, and alcoholic dye extraction was performed. A natural mordant was added to the solution to improve color adherence to the tissues.
Group A: The conventionally used H&E staining uses 1% Eosin Y as the counterstain. The slides were manually stained with H&E per the standard protocol.
Group B: The dried outer skin of Allium cepa was procured, dried at 60°C in an oven, and ground into a powder form using a motor-operated domestic mixer. The powder was then combined with alcohol and filtered to collect the stain.
To obtain a 20% solution of allium cepa dye, 20 g of pulverized onion skin was added to 100 ml of propranolol and stored in the dark for 24 h. The silt was removed by filtration. A concentrated aloe vera gel solution was added as an alternative to the chemical mordants typically used with synthetic stains. To prepare an aloe vera solution, 10 grams of aloe pulp from a freshly harvested leaf were finely mashed and boiled in 100 ml of distilled water until reduced by half. 10 ml of concentrated aloe pulp solution was mixed with the extracted stain. The staining protocol of Hematoxylin and Allium cepa (H&AC) followed the guidelines in Table 1.
Table 1.
Staining protocol for allium cepa and Bixa Orellana extracts
| Step | Reagent | H&AC (min) | H&BO (min) |
|---|---|---|---|
| Deparaffinization | Xylene I | 15 | 15 |
| Xylene II | 15 | 15 | |
| Rehydration | Absolute Alcohol | 5 | 5 |
| 95% Alcohol | 5 | 5 | |
| 80% Alcohol | 5 | 5 | |
| 70% Alcohol | 5 | 5 | |
| 60% Alcohol | 5 | 5 | |
| Tap Water | 10 | 10 | |
| Nuclear Staining | Hematoxylin | 7 | 7 |
| Tap Water | 5 | 5 | |
| Differentiation | 1% Acid Alcohol | 2 dips | 2 dips |
| Tap Water | 10 | 10 | |
| Cytoplasmic Staining | Allium cepa extract | 60 | – |
| Bixa Orellana extract | – | 45 | |
| Tap Water | 5 | 5 | |
| Dehydration | 80% Alcohol | 2 | 2 |
| 95% Alcohol | 2 | 2 | |
| Absolute Alcohol (1st) | 2 | 2 | |
| Absolute Alcohol (2nd) | 2 | 2 | |
| Alcohol + Xylene | 5 | 5 | |
| Clearing | Xylene I | 5 | 5 |
| Xylene II | 10 | 10 | |
| Mounting | DPX Mountant | — | — |
H&BO=Hematoxylin and Bixa Orellana, H&AC=Hematoxylin and Allium cepa
Group C: Dried annatto seeds were purchased, and ten grams of dried annatto seeds were measured and soaked in 100 mL of propranolol solution for 48 h to extract a 10% solution of Bixa Orellana seed stain. The stain was prepared in the same way as that for Allium cepa. The staining protocol of hematoxylin and Bixa Orellana (H&BO) followed the guidelines in Table 1.
A total of seventy-five samples were collected. The samples included 15 sections of epithelial lesions and 15 sections each of connective tissue lesions, including collagen fibers, adipose tissue, salivary gland, and bone. The samples were divided into three groups based on the cytoplasmic stains used, one each for H&E (Group A), hematoxylin and Allium cepa (Group B), and hematoxylin and Bixa Orellana (Group C), resulting in a total sample size of 225. Two histopathologists evaluated the slides to avoid interobserver bias, and the average was calculated to assess the staining parameters.
The stained slides were subsequently evaluated based on the specified parameters: stain intensity, clarity, and uniformity, and a corresponding grade was assigned. The durability and economic feasibility of the stains were also evaluated. The scoring criteria are presented in Table 2.
Table 2.
Scoring system
| Criteria | Score and grade | Description |
|---|---|---|
| Staining Intensity (I) (Assessed based on ability to observe nucleus, cytoplasm, and cell membrane) | Score 1: P (Poor) | Tissue structure is complex to appreciate (less than two of the cell structures are observed) |
| Score 2: G (Good) | Adequate tissue structure appreciation (at least two structures are observed) | |
| Score 3: E (Excellent) | A fine appreciation of tissue structure (three or more are observed) | |
| Clarity (C) | Score 1: P (Poor) | Uneven or patchy staining and lack of detail result in inadequate structure visualization |
| Score 2: G (Good) | Generally even staining, with minor variations, structures are clear and well-defined with minimal inaccuracy | |
| Score 3: E (Excellent) | Evenly distributed staining with sharply defined structures and high resolution | |
| Uniformity (U) | Score 1: P (Poor) | Patchy or uneven staining |
| Score 2: G (Good) | The stain penetrated adequately with minimal color variations | |
| Score 3: E (Excellent) | Stain penetrated tissues evenly, resulting in consistent color |
The overall efficacy of each stain (H&E, H&AC, and H&BO) was determined by taking an average of each slide’s intensity, clarity, and uniformity scores (1–3 scale), and normalizing to a 0%–100% scale for easy comparison. Stains were ranked based on the overall efficacy, identifying the most reliable staining method.
The stain’s durability was assessed over 12 months, and a score of 1 (durable slides/not faded slides) or 0 (nondurable/faded slides) was assigned. Economic viability refers to the ability of a process to be cost-effective and sustainable over time. It was assessed based on the cost of materials, cost per slide, ease of preparation, stability of stain, availability of ingredients, sustainability, and overall cost-effectiveness.
Statistical analysis
After data collection, the data were coded and entered into Microsoft Excel 2021. The data were presented as proportions. The normality of data was checked by using the Shapiro–Wilk test. The proportions among the three groups were compared by using the Chi-square test. Multiple comparisons between stains were made post hoc using the Bonferroni test to determine if there was a significant difference between the scores of stains. The median values were compared by using the Kruskal–Wallis test. Multiple comparisons were made post hoc using the Dunn test to see if there was a significant difference. SPSS, Version 23, (IBM, Chicago, Illinois, USA) was used for statistical analysis. The level of significance was set at 5%.
RESULTS
This study showed that H&E stain demonstrated significantly superior staining performance compared with the natural alternatives, with 98.67% of slides exhibiting excellent intensity, 100% excellent clarity, and 97.33% excellent uniformity, all supported by high median scores of 3.0 (interquartile range [IQR] = 0) across the parameters. Hematoxylin and Bixa Orellana (H&BO) followed as a promising alternative, displaying consistent and high-quality staining with no poor scores in any category and mean values approaching those of H&E—particularly for clarity (2.80 ± 0.40) and uniformity (2.69 ± 0.46). By contrast, Hematoxylin and Allium cepa (H&AC) showed more significant variability with lower scores across all parameters, notably achieving only 56% excellent intensity and a mean intensity of 2.49 ± 0.62. These results are further visualized in Graph 1, where H&E consistently outperformed the other stains, H&BO emerges as a viable eco-friendly substitute, and H&AC remains limited by its inconsistency [Tables 3, 4 and Graph 1].
Graph 1.
The comparison of mean values of staining properties among H&E, H&AC, and H&BO. H&E = Hematoxylin and eosin, H&BO = Hematoxylin and Bixa Orellana, H&AC = Hematoxylin and Allium cepa
Table 3.
Grade-wise comparison of various staining properties observed among different stains
| Staining properties | Grading |
n (%) |
|||
|---|---|---|---|---|---|
| H&E stain (n=75)) | H&AC stain (n=75) | H&BO stain (n=75) | Total (n=225) | ||
| Intensity | Grade 1 (Poor) | 0a | 5 (6.67)a | 0a | 5 (2.22) |
| Grade 2 (Good) | 1 (1.33)a | 28 (37.33)b | 25 (33.33)b | 54 (24.0) | |
| Grade 3 (Excellent) | 74 (98.67)a | 42 (56.00)b | 50 (66.67)b | 166 (73.78) | |
| χ2 (P) | 44.36 (<0.001**) | ||||
| Clarity | Grade 1 (Poor) | 0a | 1 (1.33)a | 0a | 1 (0.44) |
| Grade 2 (Good) | 0a | 21 (28.0)b | 15 (6.67)b | 36 (16.0) | |
| Grade 3 (Excellent) | 75 (100)a | 53 (70.67)b | 60 (80.0)b | 188 (83.56) | |
| χ2 (P) | 25.53 (<0.001**) | ||||
| Uniformity | Grade 2 (Good) | 2 (2.67)a | 28 (37.33)b | 23 (30.67)b | 53 (23.56) |
| Grade 3 (Excellent) | 73 (97.33)a | 47 (62.67)b | 52 (69.33)b | 172 (76.44) | |
| χ2 (P) | 28.19 (<0.001**) | ||||
The proportions were compared using the Chi-square test; **P<0.001 highly significant; comparison between stains was made by post hoc Bonferroni test; each superscript (a and b) letter denotes proportions do not differ significantly from each other at the P<0.05. H&E=Hematoxylin and eosin, H&BO=Hematoxylin and Bixa Orellana, H&AC=Hematoxylin and Allium cepa
Table 4.
Comparison of the median value of various staining properties observed among different stains
| Staining properties | H&E stain |
H&AC stain |
H&BO stain |
P | |||
|---|---|---|---|---|---|---|---|
| Mean±SD | Median (IQR) | Mean±SD | Median (IQR) | Mean±SD | Median (IQR) | ||
| Intensity (n=75) | 2.99±0.11 | 3.0 (0) | 2.49±0.62 | 3.0 (1) | 2.67±0.47 | 3.0 (1) | <0.001** |
| Clarity (n=75) | 3.0±0 | 3.0 (0) | 2.69±0.49 | 3.0 (1) | 2.80±0.40 | 3.0 (0) | <0.001** |
| Uniformity (n=75) | 2.97±0.16 | 3.0 (0) | 2.63±0.49 | 3.0 (1) | 2.69±0.46 | 3.0 (1) | <0.001** |
**P<0.001, highly significant. The median values compared by using the Kruskal–Wallis test. IQR=Inter-quartile range, SD=Standard deviation, H&E=Hematoxylin and eosin, H&BO=Hematoxylin and Bixa Orellana, H&AC=Hematoxylin and Allium cepa
The results of post hoc Dunn tests [Table 5] used to compare staining performance (intensity, clarity, and uniformity) between the three staining methods, H&E, H&AC, and H&BO, showed significant differences between H&E and both natural stains across all parameters (P < 0.001), confirming H&E’s superior staining quality. H&BO performed better than H&AC and is significantly closer to H&E in clarity (P = 0.003). However, there is no significant difference between H&BO and H&AC in intensity, clarity, or uniformity, indicating comparable overall efficacy between the two natural stains.
Table 5.
Multiple comparisons between stains based on the staining properties
| Multiple comparison | Intensity | Clarity | Uniformity |
|---|---|---|---|
| H&E stain versus H&AC stain | <0.001** | <0.001** | <0.001** |
| H&E stain versus H&BO stain | <0.001** | 0.003* | <0.001** |
| H&AC stain versus H&BO stain | 0.28 | 0.35 | 1.0 |
**P<0.001 is highly significant. Multiple comparisons were made using the post hoc Dunn test. H&E=Hematoxylin and eosin, H&BO=Hematoxylin and Bixa Orellana, H&AC=Hematoxylin and Allium cepa
The box plot (box-and-whisker plot) [Graph 2] visually represents the distribution of staining properties (Intensity, Clarity, Uniformity) for the three staining techniques (H&E, H&AC, and H&BO). Each box represents the IQR, showing the spread of data between the 25th and 75th percentiles. H&E stain showed a very tight distribution, with median values at the highest possible grade (3.0), indicating high consistency and superior performance. H&AC stain has a broader range, with variability in its lower whiskers, indicating inconsistent staining quality. H&BO stain has a more stable distribution than H&AC, with its median values close to the excellent range, showing it performs relatively well.
Graph 2.
Comparison of staining properties observed among different stains
The H&E stain demonstrated excellent and highly consistent staining intensity across nearly all tissue types. Epithelium, collagen, and salivary gland achieved 100% efficacy, indicating optimal staining results with strong contrast and visibility. Adipose tissue and salivary gland showed a minor deviation, with 98.52% and 99.26% efficacy, still reflecting a high staining standard. Depending on the tissue type, the H&AC stain showed noticeable variability in staining efficacy. The epithelium and bone were the most inconsistent, with an overall efficacy of 80% and 82.22%, indicating weak staining performance. Collagen, adipose tissue, and salivary glands performed relatively better, with an overall efficacy of 89.63%, 92.59%, and 89.63%. The H&BO stain provided relatively consistent and high staining intensity across all tissue types, with no poor intensity observed. Collagen stands out, with 95.56% overall efficacy. The salivary gland also showed an excellent staining efficacy with 94.07%. Epithelium, adipose tissue, and bone showed better performance than allium cepa, with values ranging from 85.19% to 89.89%. While H&BO does not reach the uniform excellence of H&E, it avoids the inconsistency seen in H&AC and maintains a dependable staining standard [Table 6 and Graph 3].
Table 6.
Overall efficacy of each tissue by stains
| Tissue | H&E efficacy (%) | H&AC efficacy (%) | H&BO efficacy (%) |
|---|---|---|---|
| Epithelium (E) | 100 | 80 | 88.89 |
| Collagen (C) | 100 | 89.63 | 95.56 |
| Adipose Tissue (AT) | 98.52 | 92.59 | 89.63 |
| Salivary Gland (SG) | 100 | 89.63 | 94.07 |
| Bone (B) | 99.26 | 82.22 | 85.19 |
H&E=Hematoxylin and eosin, H&BO=Hematoxylin and Bixa Orellana, H&AC=Hematoxylin and Allium cepa
Graph 3.
Overall efficacy percentage of each tissue among different stains
The graph [Graph 4] revealed that the H&E stain exhibits the highest overall efficacy (99.56%), reinforcing its status as the gold standard in histopathological staining. Among the natural alternatives, Hematoxylin and Bixa Orellana (H&BO) demonstrated strong and consistent performance (90.67%), making them a promising eco-friendly substitute. In contrast, despite being the most economical option, Hematoxylin and Allium cepa (H&AC) showed relatively lower efficacy (86.81%), indicating variability in staining performance. The overall efficacy scores for the three stains across different tissues indicate that H&E is the most effective stain, followed by H&BO, while H&AC showed the lowest efficacy. The horizontal bar graph illustrated the durability of three stains—H&E, H&AC, and H&BO—over 12 months, based on whether fading was present or not in 75 stained slides.
Graph 4.
Overall efficacy of stains calculated from their performance across five tissue types
H&E stain showed 100% durability, with all 75 slides (blue bar) maintaining their original intensity and no fading observed (0 orange bar). This indicates exceptional long-term stability and makes H&E the most reliable stain for archival use.
H&AC stain demonstrated moderate durability, with 57 slides (76%) showing no fading and 18 slides (24%) showing fading. The presence of a noticeable orange segment highlights its lesser stability compared with the other stains.
H&BO stain performed well, with 69 slides (92%) not fading and only six slides (8%) showing fading. While not as flawless as H&E, H&BO still offers strong durability, making it a dependable choice for long-term storage with minimal degradation [Table 7 and Graph 5].
Table 7.
Durability of the stains over a period of 12 months
| Durability | H&E (per 75 slides) | H&AC (per 75 slides) | H&BO (per 75 slides) |
|---|---|---|---|
| Fading not present (1) | 75 | 57 | 69 |
| Fading present (0) | 0 | 18 | 6 |
H&E=Hematoxylin and eosin, H&BO=Hematoxylin and Bixa Orellana, H&AC=Hematoxylin and Allium cepa
Graph 5.
Durability of three stains over 12 months
Economic viability inference
The organic stains, H&AC and H&BO, presented promising, eco-friendly alternatives with strong economic appeal. H&AC is the most cost-effective stain at ₹9.9 per slide, ideal for budget-friendly use. H&BO offered a good balance at ₹12.8, while H&E is the costliest at ₹14.4 per slide. Although H&E is more stable (lasting 3–6 months) and easier to prepare, it is carcinogenic. H&AC and H&BO are biodegradable, nontoxic, and reduce environmental and regulatory concerns. While they have a shorter shelf life than H&E, their ease of preparation and sustainability make them excellent options for institutions aiming to adopt greener, safer histological practices.
DISCUSSION
Hematoxylin and eosin continue to be the benchmark in histopathological staining. This study comprehensively evaluated the overall staining efficacy of Hematoxylin and Eosin, Hematoxylin and Allium cepa, and Hematoxylin and Bixa Orellana by assessing intensity, clarity, uniformity, economic feasibility, and durability across five tissue types. The results indicated that H&E staining consistently outperformed the alternative stains in all significant parameters assessed, with the mean values of intensity, clarity, and uniformity being 2.99 ± 0.11, 3.0 ± 0, and 2.97 ± 0.16, respectively. The findings were in agreement with the observations of Navya et al., Sarode et al., and Uchejeso et al. who found that H&E maintained the highest scores for cytoplasmic visualization, nuclear detail, and overall tissue architecture in tissue samples.[2,3,4]
Previous studies (Padhi, Bordoloi et al., Bose et al., and Adnan et al.) have highlighted concerns about the environmental toxicity of synthetic dyes, particularly eosin Y, which is classified as a Group 3 carcinogen by the IARC. It is toxic upon ingestion or inhalation and may cause allergic reactions such as erythema, pruritus, and inflammation, along with ecological harm. Despite its superior staining properties, the push for eco-friendly alternatives has led to the exploration of organic dyes.[9,10,11,12]
Prior phytochemical studies supported the rationale for selecting the organic sources in our research. Active colorants such as flavonoids, phenols, carotenoids, and glycosides—identified in natural sources like roselle, alkanet, and pomegranate flowers—are known to interact with tissue proteins through mechanisms including hydrogen bonding and metal chelation, as demonstrated by Avwioro et al., Saiki et al., Nilgün et al.[13,14,15] Their interaction with proteins in tissues—often through hydrogen bonding or metal chelation—accounted for their tissue-specific affinities.
In line with these established protocols, our study employed ethanolic extraction of onion peel and annatto seeds to standardize the extraction of natural stains. Supporting this approach, previous research by Avwioro et al., Sridhara et al., Pereira et al., and Smith-Togobo et al.[13,16,17,18] has emphasized that ethanol enhanced the solubility of pigment compounds and facilitated their penetration into tissue matrices, thereby directly influencing staining efficacy.
This study investigated the use of dried red onion skin and annatto seed extracts. To our knowledge, annatto-based staining of human histopathological sections is novel. Hematoxylin and Bixa Orellana emerged as a promising alternative, closely followed by Hematoxylin and Allium cepa. The respective mean values of intensity, clarity, and uniformity were: Bixa Orellana (2.67 ± 0.47, 2.80 ± 0.40, and 2.69 ± 0.46) and Allium cepa (2.49 ± 0.62, 2.69 ± 0.49, and 2.63 ± 0.49). This contrasts with Nnaemeka et al.’s study conducted in rats, which found Bixa ineffective in peripheral blood smears.[19]
In contrast, Suryawanshi et al. reported that curcumin-based stains underperformed in bone tissue.[1] Allium cepa was more effective than annatto for adipose tissue among the organic stains.
In tissue-specific analysis, H&E provided excellent results for epithelium, collagen, and salivary gland, and near-excellent results for adipose tissue and bone (98.52%–100%). H&BO yielded promising results for collagen and salivary glands, with near-excellent results for epithelium, adipose tissue, and bone (85.19%–95.56%). Comparable findings were reported by Shehu et al. and Nilgun et al., who demonstrated that the intensity of cytoplasmic and nuclear staining obtained using Cola nut and Punica granatum flower extracts was comparable to eosin.[15,20] The staining efficacy across tissue using H&AC ranged between 80% and 92.59%, with adipose tissue showing the best results and epithelium the least. These findings align with Itodo et al. and Krampah et al., who observed excellent cytoplasmic and connective tissue staining using Allium cepa skin extracts in human and animal tissues.[7,21] By contrast, Suryawanshi et al. reported that curcumin-based stains underperformed in bone tissue.[1] Allium cepa was more effective than annatto for adipose tissue among the organic stains [Figure 1].
Figure 1.
(a-c) Epithelium in ×10 view in H&E, H&AC, and H&BO; (d-f) Collagen fibers in ×10 view in H&E, H&AC, and H&BO; (g-i) Adipose tissue in ×10 view in H&E, H&AC, and H&BO; (j-l) Salivary gland in ×10 view in H&E, H&AC, and H&BO; and (m-o) Bone in ×10 view in ×10 view in H&E, H&AC, and H&BO. H&E = Hematoxylin and eosin, H&BO = Hematoxylin and Bixa Orellana, H&AC = Hematoxylin and Allium cepa
In addition, studies by Avwioro et al., Raheem et al., C. Ceccopieri et al., Sarode et al., and Prajapati et al. have identified several natural alternatives to eosin, such as Pterocarpus osun, Hibiscus Sabdariffa, Crocus sativus flowers, food coloring agents, Zingiber officinale, and Curcuma longa.[3,13,22,23,24]
From a cost and sustainability perspective, H&AC was the most economical at ₹9.9 per slide, with H&BO being moderately priced (₹12.8 per slide) and H&E the most expensive (₹14.4 per slide). H&AC and H&BO are eco-friendly and noncarcinogenic, supporting the shift toward sustainable laboratory practices. However, their limited shelf life (1 week) compared with H&E (3–6 months) poses a logistical challenge.
Durability testing over 12 months showed that H&E slides retained complete staining integrity with no fading, while H&BO showed 92% durability, and H&AC lagged with 76% durability from total slides stained. Sarode et al. reported similar results when studying food coloring agents as safer alternatives to eosin.[3]
CONCLUSIONS
While H&E remains the most robust stain regarding diagnostic utility, stability, and archival integrity, H&BO offers a viable, eco-friendly, and moderately effective alternative suitable for short-term applications and settings with budget constraints. Despite its affordability and sustainability, H&AC is limited by its inconsistency and reduced staining efficacy. Future research should explore optimized formulations of alternative stains, incorporating stabilizers to enhance longevity without compromising environmental safety or diagnostic accuracy.
Conflicts of interest
There are no conflicts of interest.
Acknowledgement
We gratefully acknowledge the Department of Oral Pathology for their invaluable support and allowing us to undertake this research.
Funding Statement
Nil.
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